7.4: DNA Repair - Biology

7.4: DNA Repair - Biology

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Source: BiochemFFA_7_3.pdf. The entire textbook is available for free from the authors at

Safeguarding the genome

In the last section we considered the ways in which cells deal with the challenges associated with replicating their DNA, a vital process for all cells. It is evident that if DNA is the master copy of instructions for an organism, then it is important not to make mistakes when copying the DNA to pass on to new cells. Although proofreading by DNA polymerases greatly increases the accuracy of replication, there are additional mechanisms in cells to further ensure that newly replicated DNA is a faithful copy of the original, and also to repair damage to DNA during the normal life of a cell.

DNA damage

All DNA suffers damage over time, from exposure to ultraviolet and other radiation, as well as from various chemicals in the environment (Figures 7.34 & 7.35). Even chemical reactions naturally occurring within cells can give rise to compounds that can damage DNA. As you already know, even minor changes in DNA sequence, such as point mutations can sometimes have far-reaching consequences. Likewise, unrepaired damage caused by radiation, environmental chemicals or even normal cellular chemistry can interfere with the accurate transmission of information in DNA. Maintaining the integrity of the cell's "blueprint" is of vital importance and this is reflected in the numerous mechanisms that exist to repair mistakes and damage in DNA.

Post-replicative mismatch repair

We earlier discussed proofreading by DNA polymerases during replication. While proofreading significantly reduces the error rate, not all mistakes are fixed on the fly by DNA polymerases.

What mechanisms exist to correct the replication errors that are missed by the proof-reading function of DNA polymerases? Errors that slip by proofreading during replication can be corrected by a mechanism called mismatch repair. While the error rate of DNA replication is about one in 107 nucleotides in the absence of mismatch repair, this is further reduced a hundred-fold to one in 109 nucleotides when mismatch repair is functional.

What are the tasks that a mismatch repair system faces?

It must:​

  • Scan newly made DNA to see if there are any mispaired bases (e.g., a G paired to a T)​
  • Identify and remove the region of the mismatch.​
  • Correctly fill in the gap created by the excision of the mismatch region.

Distinguishing strands

Importantly, the mismatch repair system must have a means to distinguish the newly made DNA strand from the template strand, if replication errors are to be fixed correctly. In other words, when the mismatch repair system encounters an A-G mispair, for example, it must know whether the A should be removed and replaced with a C or if the G should be removed and replaced with a T.

But how does the mismatch repair system distinguish between the original and the new strands of DNA? In bacteria, the existence of a system that methylates the DNA at GATC sequences is the solution to this problem. E.coli has an enzyme, DNA adenine methylase (Dam) that adds methyl groups on the to adenines in GATC sequences in DNA (Figure 7.36). Newly replicated DNA has not yet undergone methylation and thus, can be distinguished from the template strand, which is methylated.

The mismatch repair proteins selectively replace the strand lacking methylation, thus ensuring that it is mistakes in the newly made strand that are removed and replaced. Because methylation is the criterion that enables the mismatch repair system to choose the strand that is repaired, the bacterial mismatch repair system is described as being methyl-directed.

Figure 7.36- Dam methylase adds methyl groups at GATC sequences

Mismatch repair genes

Mismatch repair has been well studied in bacteria, and the proteins involved have been identified. In E.coli, mismatch repair proteins are encoded by a group of genes collectively known as the mut genes. Important components of the mismatch repair machinery are the proteins MutS, MutL and MutH (Figure 7.37).

MutS acts to recognize the mismatch, while MutL and MutH are recruited to the mismatch site by the binding of MutS. MutH is an endonuclease that cuts the newly synthesized and, as yet, unmethylated DNA strand at a GATC. This activates a DNA helicase and an exonuclease that help unwind and remove the region containing the mismatch. DNA polymerase III fills in the gap, using the opposite strand as the template, and ligase joins the ends, to restore a continuous strand.

Eukaryotes also have a mismatch repair system that repairs not only single base mismatches but also insertions and deletions. Homologs to the E. coli MutS and MutL have been identified in other organisms, including humans: hMSH1 and hMSH2 (human MutS homolog 1 and 2) are homologous to MutS, while hMLH 1 is homologous to MutL. These, together with additional proteins, carry out mismatch repair in eukaryotic cells.

DNA methylation is not used by eukaryotic cells as a way to distinguish the new strand from the template, and it is not yet completely understood how the mismatch repair system in eukaryotes "knows" which strand to repair. There is evidence that the newly made DNA may be recognized by the fact that it is nicked, or discontinuous. This suggests that discontinuity resulting from Okazaki fragments that have not yet been joined together may permit the new strand to be distinguished from the old, continuous template strand.

Repairing damage to DNA

In the preceding section we looked at mistakes made when DNA is copied, where the wrong base is inserted during synthesis of the new strand. But even DNA that is not being replicated can get damaged or mutated. These sorts of damage are not associated with DNA replication, rather they can occur at any time.

What causes damage to DNA? ​
Some major causes of DNA damage are:​
a. Radiation (e.g., UV rays in sunlight and in tanning booths, or ionizing radiation)​
b. Exposure to damaging chemicals, such as nitrosamines or polycyclic aromatic hydrocarbons, in the environment (see Figure 7.38)​
c. Chemical reactions within the cell (such as the deamination of cytosine to give uracil, or the methylation of guanine to produce methylguanine).

This means the DNA in your cells is vulnerable to damage simply from normal sorts of actions, such as walking outdoors, being in traffic, or from the chemical transformations occurring in every cell as part of its everyday activities. (Naturally, the damage is much worse in situations where exposure to radiation or damaging chemicals is greater, such as when people use tanning beds, or smoke, regularly.)

Types of damage

What kinds of damage do these agents cause? Radiation can cause different kinds of damage to DNA.

Sometimes, as with much of the damage done by UV rays, two adjacent pyrimidine bases in the DNA will be cross-linked to form cyclobutane pyrimidine dimers or CPDs (see Figure 7.39). Note that these are two neighboring pyrimidine bases on the same strand of DNA. UV exposure can also lead to the formation of another type of lesion, known as a (6-4) photoproduct or 6-4PP (Figure 7.39). Ionizing radiation can cause breaks in the DNA backbone, in one or both strands.

Figure 7.39 - Possible chemical structures of a pyrimidine dimer - 6-4PP (left) and CPD (right) - Wikipedia

Molecules like benzopyrene, found in automobile exhaust, can attach themselves to bases, forming bulky DNA adducts in which large chemical groups are linked to bases in the DNA. Damage like pyrimidine dimers, 6-4PPs or chemical adducts can physically distort the DNA helix, causing DNA and RNA polymerase to stall when they attempt to copy those regions of DNA (Figure 7.40).

Chemical reactions occurring within cells can cause cytosines in DNA to be deaminated to uracil. Other sorts of damage in this category include the formation of oxidized bases like 8-oxo-guanine or alkylated bases like O6-methylguanine. These do not actually change the physical structure of the DNA helix, but they can cause problems because uracil and 8-oxo-guanine pair with different bases than the original cytosine or guanine, leading to mutations on the next round of replication. O6-methylguanine similarly can form base pairs with thymine instead of cytosine.

Removing damage

Cells have several ways to remove the sorts of damage described above. The first of these is described as direct reversal. Many organisms (though, unfortunately for us, not humans) can repair UV damage like CPDs and 6-4PPs because they possess enzymes called photolyases (photo=light; lyase=breakdown enzyme - Figure 7.41). Photolyases work through a process called photoreactivation, and use blue light energy to catalyze a photochemical reaction that breaks the aberrant bonds in the damaged DNA and returns the DNA to its original state.

Suicide enzyme

O6-methylguanine in DNA can also be removed by direct reversal, with the help of the enzyme O6-methylguanine methyltransferase. This is a very unusual enzyme that removes the methyl group from the guanine and transfers it onto a cysteine residue in the enzyme. The addition of the methyl group to the cysteine renders the enzyme non-functional.

As you know, most enzymes are catalysts that remain unchanged over the course of the reaction, permitting a single enzyme molecule to repeatedly catalyze a reaction. Because the O6-methylguanine methyltransferase does not fit this description, it is sometimes not regarded as a true enzyme. It has also been called a suicide enzyme, because the enzyme “dies” as a result of its own activity.

Excision repair

Excision repair is another common strategy. Excision repair is a general term for the cutting out and re-synthesizing of the damaged region of a DNA. There are several different kinds of excision repair, but they all involve excising the portion of the DNA that is damaged, followed by repair synthesis using the other strand as template, and finally, ligation to restore continuity to the repaired strand. Cells possess several different kinds of excision repair, each geared to specific kinds of DNA damage. Between them, these repair systems deal with the wide variety of insults to the genome.

Nucleotide excision repair

Nucleotide excision repair (NER) fixes damage such as the formation of chemical adducts, as well as UV damage. Both chemical adducts and the formation of CPDs or 6,4 photoproducts can cause significant distortion of the DNA helix. NER proteins act to cut the damaged strand on either side of the lesion. A short portion of the DNA strand containing the damage is then removed and a DNA polymerase fills in the gap with the appropriate nucleotides. Nucleotide excision repair has been extensively studied in bacteria.

In E. coli, recognition and excision of the damage is carried out by a group of proteins encoded by the uvrABC and uvrD genes. The protein products of the uvrA, uvrB and uvrC genes function together as the so-called UvrABC excinuclease. The damage is initially recognized and bound by a complex of the UvrA and UvrB proteins. Once the complex is bound, the UvrA dissociates, leaving the UvrB attached to the DNA, where it is then joined by the UvrC protein.

Strand nicking

It is the complex consisting of UvrB and C that acts to cut the phosphodiester backbone on either side of the damage, creating nicks in the strand about 12-13 nucleotides apart. A helicase encoded by uvrD then unwinds the region containing the damage, displacing it from the double helix together with UvrBC. The gap in the DNA is filled in by DNA polymerase, which copies the undamaged strand, and the nick is sealed with the help of DNA ligase.

Nucleotide excision repair is also an important pathway in eukaryotes. It is particularly important in the removal of UV damage in humans, given that we lack photolyases. A number of proteins have been identified that function in ways similar to the Uvr proteins.

The importance of these proteins is evident from the fact that mutations in the genes that encode them can lead to a number of genetic diseases, like Xeroderma pigmentosum, or XP. People with XP are extremely sensitive to UV exposure, because the damage caused by it cannot be repaired, leaving them at a much higher risk of developing skin cancer.

Two repair modes

Nucleotide excision repair operates in two modes, one known as global genomic repair and the other as transcription-coupled repair. While the function of both is to remove helix-destabilizing damage like cyclobutane pyrimidine dimers or chemical adducts, the way in which the lesions are detected differs.

In global genomic repair, damage is identified by surveillance of the entire genome for helix distorting lesions. In the case of transcription-coupled repair, the stalling of the RNA polymerase at a site of DNA damage is the indicator that activates this mode of nucleotide excision repair.

Base excision repair

Base excision repair (BER) is a repair mechanism that deals with situations like the deamination of cytosine to uracil (Figure 7.43) or the methylation of a purine base. These changes do not typically distort the structure of the DNA helix, unlike chemical adducts or UV damage.

In base excision repair a single damaged base is first removed from the DNA, followed by removal of a region of the DNA surrounding the missing base. The gap is then repaired.

Uracil-DNA glycosylase

The removal of uracil from DNA is accomplished by the enzyme uracil-DNA glycosylase that can recognize uracil in DNA and break the glycosidic bond between the uracil and the sugar in the nucleotide (Figure 7.44). The removal of the base leaves a gap called an apyrimidinic site (AP site) because, in this case, uracil, a pyrimidine was removed. It is important to remember that at this point the backbone of the DNA is still intact, and the removal of a single base simply creates a gap like a tooth that has been knocked out.

The formation of the AP site triggers the activity of an enzyme known as an AP endonuclease that cuts the DNA backbone 5’ to the AP site. In the remaining steps, a DNA polymerase binds to the nick, then using its exonuclease and polymerase activities, replaces the sequence in this region. Depending on the situation, a single nucleotide may be replaced (short patch BER) or a stretch of several nucleotides may be removed and replaced (long patch BER). Finally, as always, DNA ligase acts to seal the nick in the DNA.

Repair of double-strand breaks

While all the repair mechanisms discussed so far fixed damage on one strand of DNA using the other, undamaged strand as a template, these mechanisms cannot repair damage to both strands. What happens if both strands are damaged? Ionizing radiation, exposure to certain chemicals, or reactive oxygen species generated in the cell can lead to double-strand breaks (DSBs) in DNA.

DSBs are a potentially lethal form of damage that, in addition to blocking replication and transcription, can also lead to chromosomal translocations, where part of one chromosome gets attached to a piece of another chromosome. Two different cellular mechanisms exist that help repair DSBs (Figure 7.45), homologous recombination (HR) and non-homologous end joining (NHEJ).

Figure 7.45 - Non-homologous end joining (left) versus homologous recombination (right) - Wikipedia

Homologous recombination repair commonly occurs in the late S and G2 phases of the cell, when each chromosome has been replicated and information from a sister chromatid can be used as a template to achieve error-free repair. Note that in contrast to excision repair, where the damaged strand was removed and the undamaged sister strand served as the template for filling in the damaged region, HR must use the information from another DNA molecule, because both strands of the DNA are damaged in DSBs.

Nuclease action

Detection of the double-strand break triggers nuclease activity that chews back one strand on each end of the break. This results in the production of single-stranded 3’ overhangs on each end. These single-stranded ends are bound by several proteins, creating a nucleoprotein filament that can then “search” for homologous (matching) sequences on a sister chromatid.

When such sequences are found, the nucleoprotein filament invades the undamaged sister chromatid, forming a crossover. This creates heteroduplexes made up of DNA strands from different chromatids. Strand invasion (Figure 7.47) is followed by branch migration, during which the Holliday junction moves along the DNA, extending the heteroduplex away from the original site of the crossover (Figure 7.48). In E. coli, branch migration depends on the activity of two proteins, RuvA and RuvB. The resulting recombination intermediate can be resolved, with the help of RuvC to give complete, error-free strands.

Non-homologous end joining

In contrast to homologous recombination, Non-homologous end joining (NHEJ) is error-prone. It does not use or require a homologous template to copy, and works by simply chewing back the broken ends of DSBs and ligating them together. Not surprisingly, NHEJ introduces deletions in the DNA as a result.

Translesion DNA synthesis

As we have seen, cells have a variety of mechanisms to help safeguard the integrity of the information in DNA. One measure of last resort is translesion DNA synthesis, also known as bypass synthesis. Translesion synthesis occurs when a DNA polymerase encounters DNA damage on the template strand, but instead of stalling or skipping past the damage, replication switches to an error-prone mode, ignoring the template and incorporating random nucleotides into the new strand. In E.coli, translesion synthesis is dependent on the activities of proteins encoded by the umuC and umuD genes. Under the appropriate conditions (see SOS response, below) UmuC and UmuD are activated to begin bypass synthesis. Being error-prone, translesion synthesis gives rise to many mutations.

The SOS response

Named for standard SOS distress signals, the term “SOS repair” refers to a cellular response to UV damage. When bacterial cells suffer extensive damage to their DNA as a result of UV exposure, they turn on the coordinated expression of a large number of genes that are necessary for DNA repair. These include the uvr genes needed for nucleotide excision repair and recA, which is involved in homologous recombination. In addition to these mechanisms, which can carry out error-free repair, the SOS response can also induce the expression of translesion polymerases encoded by the dinA, dinB and umuCD genes.

How are all these genes induced in a coordinated way following UV damage? All of the genes induced in the SOS response are regulated by two components. The first is the presence of a short DNA sequence upstream of their coding region, called the SOS box. The second is a protein, the LexA repressor (Figure 7.49), that binds to the SOS box and prevents transcription of the downstream genes. Expression of the genes requires the removal of LexA from its binding site. How is this achieved?

When exposure to radiation results in DNA breaks, the presence of single-stranded regions triggers the activation and binding of RecA proteins to the single-stranded region, creating a nucleoprotein filament. The interaction of the RecA with the LexA repressor leads to autocleavage of the repressor, allowing the downstream gene(s) to be expressed (Figure 7.50).

The genes controlled by the LexA repressor, as mentioned earlier, encode proteins that are necessary for accurate DNA repair as well as error-prone translesion synthesis. The various genes involved in DNA repair are induced in a specific order. In the initial stages, the repair genes that are derepressed are for nucleotide excision repair, followed by homologous recombination, both error-free mechanisms for repair. If the damage is too extensive to be repaired by these systems, error-prone repair mechanisms may be brought into play as a last resort.

SOS response and antibiotic resistance

The increased mutation rate in the SOS response may play a role in the acquisition of antibiotic resistance in bacteria (Figure 7.51).

An example is the development of resistance to topoisomerase poisons like the fluoroquinolone family of drugs. Fluoroquinolones inhibit the ability of topoisomerases to religate the ends of their substrates after nicking them to allow overwound DNA to relax. This results in accumulation of strand breaks that can trigger the SOS response. As a consequence of error-prone DNA synthesis by low fidelity polymerases during the SOS response, there is a large increase in the number of mutations. While some mutations may be lethal to the bacteria, others can contribute to the rapid development of drug resistance in the population.

Section Summary

DNA polymerase can make mistakes while adding nucleotides. It edits the DNA by proofreading every newly added base. Incorrect bases are removed and replaced by the correct base, and then a new base is added. Most mistakes are corrected during replication, although when this does not happen, the mismatch repair mechanism is employed. Mismatch repair enzymes recognize the wrongly incorporated base and excise it from the DNA, replacing it with the correct base. In yet another type of repair, nucleotide excision repair, the incorrect base is removed along with a few bases on the 5′ and 3′ end, and these are replaced by copying the template with the help of DNA polymerase. The ends of the newly synthesized fragment are attached to the rest of the DNA using DNA ligase, which creates a phosphodiester bond.

Most mistakes are corrected, and if they are not, they may result in a mutation defined as a permanent change in the DNA sequence. Mutations can be of many types, such as substitution, deletion, insertion, and translocation. Mutations in repair genes may lead to serious consequences such as cancer. Mutations can be induced or may occur spontaneously.

A newly discovered protein repairs DNA

Credit: Pixabay/CC0 Public Domain

Researchers from the University of Seville, in collaboration with colleagues from the Universities of Murcia and Marburg (Germany) have identified a new protein that makes it possible to repair DNA. The protein in question, called cryptochrome, has evolved to acquire this and other functions within the cell.

Ultraviolet radiation can damage the DNA, leading to mutations that disrupt cell function and can allow cancer cells to grow out of control. Our cells have DNA repair systems to defend themselves against this sort of damage. One of these systems is based on a protein, photolyase, which uses blue light to repair DNA damage before it leads to mutations.

Over the course of evolution, the genes for photolyase duplicated and became specialized, creating new proteins, cryptochromes, which have honed their ability to perceive blue light and now perform other functions in cells. For example, cryptochromes use blue light as a signal to regulate plant growth and the rhythm that controls daily activity (the circadian rhythm) in fungi and animals.

The authors of this study discovered that in the fungus Mucor circinelloides, a human pathogen, cryptochromes are the protein responsible for DNA repair after exposure to ultraviolet radiation, a function that should be performed by photolyase. They also suggest that cryptochromes in this fungus acquired their ability to repair DNA during evolution from an ancestral cryptochrome that was not able to repair DNA. This discovery illustrates how proteins change as their functions evolve.

What is DNA repair?

DNA polymerase enzyme sometimes accidentally introduces wrong bases which will disrupt the normal Watson-Crick base paring of the DNA. There are also many possibilities of DNA damage during genetic recombination happens during gametogenesis by meiotic cell division. If the damages or errors in the DNA are not corrected in the somatic cells, it may leads to the development of cancer or it results in the loss of function of genes. More than that, if DNA damages occur in the gametes is not rectified, it will be carried over to next generation through progenies. Thus, damage to the genetic materials is a major threat to all organisms. In order to counteract these threats, cells has evolved many methods to overcome and rectify different types DNA damages. All these methods are collectively termed as DNA REPAIR mechanisms. Similar to DNA replication, transcription and translation, the process of DNA repair is also a prime molecular event in the cells which is very essential for the ultimate survival of the cells and also for the survival of the organism.

DNA Repair and Nobel Prize in Chemistry (2015)

The Royal Swedish Academy of Sciences awarded the 2015 Nobel Prize in Chemistry for the discovery and contributions of DNA repair mechanism. The Nobel Prize in Chemistry this year was shared by three scientists namely Thomas Lindhal, Paul Modrich and Aziz Sancar for their “Mechanistic studies of DNA repair”. The detailed mechanism of DNA repair in the cells that we know today is primarily due to their research. Professor Thomas Lindahl demonstrated that the DNA is an unstable molecule which is subjected to damage even under physiological conditions. He also identified a completely new DNA glycosylase enzyme and described their role DNA repair mechanisms. Professor Paul Modrich transformed the field of mismatch repair to a detailed biochemical understanding first in bacteria and later in eukaryotes. Professor Sancar explained the mechanism of nucleotide excision repair first in bacteria and later in eukaryotic cells. He also explained the molecular mechanisms behind the photoreactivation process, which is type of light dependent DNA repair mechanism. All these contributions helped us to understand the nature of some diseases like cancer and they helped to develop new therapies against many diseases including cancer.

Destructive forces faced by DNA in the cell

There are two categories of destructive forces in the cells that could damage the DNA both structurally and chemically. They are:

(1). Internal or intrinsic factors: they includes:-

Ø Metabolic intermediates

(2). External or extrinsic factors: they includes:-

Ø Radiations (X-rays, UV rays, γ-rays)

Ø Carcinogens/ DNA intercalating agents

As the name suggests, internal factors originate inside the cell itself. Reactive free radicals and metabolic intermediates or metabolic byproducts can severely damage DNA and can induce spontaneous mutations. For example, the oxidative deamination of nucleotides in the cells which results in the conversion of cytosine to uracil is caused by metabolic intermediates. Errors happen during DNA replication and recombination are also considered as the internal factors.

Major external factors that could damage cellular DNA fall under two sub-categories. Among which the different types of radians are most important one. Radiations like UV rays, X-rays and γ-rays can severely disrupt the structure and chemistry of DNA leading to a variety of mutation possibilities. Similarly different carcinogenic chemicals, which we generally called as DNA intercalating agents, can directly react with DNA and can cause different structural and chemical modifications in the DNA.

When the normal conformational chemistry of DNA is lost due to any of these internal or external factors, we can say that there is a lesion in the DNA. As in the image, the normal conformational symmetry of DNA is lost due to formation of thymine dimer by UV Light and this created a bulge in one strand. The bulge produced by thymine dimer can be called as DNA lesion.

The possible structural lesions that can happens to DNA:

(1). Thymine dimer formation:

The most common structural lesion in the DNA is the formation of pyrimidine dimer. It is formed by the covalent bond formation between two adjacent pyrimidine residues such as between two thymine or two cytosine or very rarely a thymine and a cytosine. Pyrimidine dimer formation is caused by the ultraviolet irradiation of DNA. Among the three types of pyrimidine dimers, the thymine dimer formation is the most common one. When DNA is struck with UV light, the hydrogen bonds between the two strands breaks and two covalent bonds are formed between the two thymine residues. The two covalent bonds are formed by the breakage of two double bonds present between C5 and C6 of adjacent thymine residues. The Thymine dimer is also called as cyclobutane photodimer or CPD since it structurally resembles cyclobutane nucleus. If thymine dimer in the DNA is left uncorrected, it will cause melanoma, which is a type of skin cancer.

(2). Spontaneous depurination of DNA

It is due to the spontaneous removal of adenine or guanine residues from the DNA due to the cleavage of N-glycosyl bond which connects the nitrogen base with the deoxyribose sugar. Depurination results in the formation of apurinic site (AP site). Apurinic site structurally disrupt the normal conformation of DNA. If apurinic site is left uncorrected, many types of cancerous growth initiates in the tissue.

(3). Spontaneous deamination of bases in the DNA

As the name suggests, it the removal of amino groups from the nitrogen bases of DNA. Deamination is usually caused by the oxidative removal of amino group from the nitrogen bases. Deamination produces unnatural bases or change in the base sequence and results in point mutation in the DNA. Unnatural bases are bases other than adenine, guanine, thymine, cytosine or urasil. Hypoxanthine and xanthine are the two unnatural bases incorporated to DNA due to spontaneous deamination.

Deamination of adenine creates hypoxanthine formation. Deamination of guanine creates another unnatural base called xanthine. Deamination of cytosine creates uracil. Since, uracil is not a DNA base it will destroy the normal Watson-Crick base pairing of the DNA. Because of the absence of amino group, the deamination of thymine residue is not possible. There is one more type of deamination, in fact it is the most severe and dangerous one. As a part of DNA regulation mechanism, most of the cytosine resides in the DNA will be methylated in its 5th position as 5 methyl cytosine. The deamination of 5-methyl cytosine produces thymine and thus it creates a point mutation.

(4). Errors in DNA replication/Recombination

This is due the accidental inclusion of wrong bases in the DNA by DNA polymerase during DNA replication. DNA polymerase also has an exonuclease activity which usually removes the wrong bases and inserts the correct base by a process called ‘proof reading’. Sometimes the proof reading method fails to detect the wrong base and the wrong base persist in the DNA. If these errors are not corrected, in the next replication cycle base change occurs in the DNA and one daughter DNA get mutated.

Different DNA repair mechanisms in the cell:

In this post, we will just mention the names of different DNA repair mechanisms we have detailed posts with video tutorials for each of these repair mechanisms.

So far there are six different types of DNA repair mechanisms known to science.

(1). Photoreactivation: a light depended DNA repair mechanism which removes thymine dimers

(2). Base Excision Repair (BER): only the damaged base is removed or excised from the DNA strand without removing the normal bases

(3). Nucleotide excision repair (NER): the damaged bases along with a short stretch of healthy stand is removed and refilled with correct bases

(4). Mismatch repair or MMR: As the name suggests it removes the mismatched bases from the DNA and fill with correct bases.

(5). Double strand break repair: double strand break lesions are rectified

(6). Homology directed repair: here a long stretch of DNA repair takes places by consulting with the sequence in the homologous chromosome

There is another type of DNA repair strategy in the cells called SOS response, is actually not a DNA repair mechanism. SOS response is initiated in the cells after a sever DNA damage. SOS responses trigger many molecular processes in the cells and DNA repair is one among these processes.

The repair mechanisms such as, photoreactivation, base excision repair, nucleotide excision repair and mismatch repair, only the damaged strand of the DNA duplex is repaired and the undamaged strand acts as the template strand. However in double strand break repair and homology directed repair, both the strands of a DNA duplex are repaired.

Lecture notes

  • these features allow tRNA to attach to binding sites on ribosomes and to mRNA
  • variable features in each type of tRNA produce different physical and chemical properties, allowing for the correct binding of amino acids to specific tRNAs
  • tRNA activating enzyme attaches a specific amino acid to the 3’ end of a tRNA
  • there are 20 different tRNA activating enzymes, one for each of the 20 amino acids
  • each of these enzymes attaches one particular amino acid to all of the tRNA molecules that have an anticodon corresponding to that amino acid
  • ATP hydrolysis provides the energy for amino acid attachment to tRNA this stored energy is also used later to link the amino acid to the growing polypeptide chain during translation

7.4.2 Outline the structure of ribosomes including:

  • protein (40% of the weight) and rRNA (60% of weight) composition:
  • large and small subunits
  • three tRNA binding sites: E-site, P-site, A-site
  • mRNA binding site: on small ribosomal subunit

7.4.3 State that translation consists of initiation, elongation, translocation and termination

7.4.4 State that translation occurs in a 5’ -> 3’ direction

7.4.5 Draw and label a diagram showing the structure of a peptide bond between two amino acids.

7.4.6 Explain the process of translation, including ribosomes, polysomes, start codons and stop codons:

Click to complete two simple interactive animations of protein synthesis:

  • 5’ end of mRNA binds to the small subunit of the ribosome
  • initial mRNA codon = AUG = start codon
  • tRNA with anticodon: UAC binds to mRNA AUG codon by complementary base pairing, methionine attached to tRNA 3’ terminal
  • large ribosomal subunit binds, completing ribosomal structure, and producing two ribosomal binds sites: P site & A site

  • tRNA with anticodon complementary to second mRNA codon binds to ribosomal A site, with appropriate amino acid attached to tRNA 3’ terminal
  • enzymes in ribosome catalyze formation of peptide bond between methionine and 2nd amino acid
  • P site tRNA, now separated from methionine, exits ribosome
  • ribosome moves one codon (3 nucleotides) toward the 3’ end of mRNA, thus shifting previous A-site tRNA to P-site, and opening A-site
  • tRNA with anticodon complementary to A-site mRNA codon binds to ribosomal A-site, with appropriate amino acid attached to tRNA 3’ terminal
  • enzymes in ribosome catalyze formation of peptide bond between 2nd and 3rd amino acids
  • P site tRNA, now separated from its amino acid, exits ribosome
  • ribosome moves one codon (3 nucleotides) toward the 3’ end of mRNA, thus shifting previous A-site tRNA to P-site, and opening A-site
  • repetition of process until stop codon is reached
  • when ribosomal A-site reaches a stop codon, no tRNA has a complementary anticodon
  • release factor protein binds to ribosomal A-site stop codon
  • polypeptide and mRNA are released
  • large and small ribosomal subunits separate

D. polysomes: several to many ribosomes translating the same mRNA into protein each moving in the 5’ to 3’ direction

E. start codon: the mRNA triplet codon AUG is universally the start codon used to mark the beginning of the coding sequence of a gene thus, the tRNA with the anticodon UAC and carrying the amino acid methionine is always the first tRNA to enter the P-site during translation

F. stop codon: there are three stop codons in the genetic code none of these have a corresponding tRNA instead, when a ribosome encounters a stop codon, a release factor binds to the stop codon, which terminates translation and allows the separation of all of its components

Section Summary

DNA polymerase can make mistakes while adding nucleotides. It edits the DNA by proofreading every newly added base. Incorrect bases are removed and replaced by the correct base, and then a new base is added. Most mistakes are corrected during replication, although when this does not happen, the mismatch repair mechanism is employed. Mismatch repair enzymes recognize the wrongly incorporated base and excise it from the DNA, replacing it with the correct base. In yet another type of repair, nucleotide excision repair, the incorrect base is removed along with a few bases on the 5′ and 3′ end, and these are replaced by copying the template with the help of DNA polymerase. The ends of the newly synthesized fragment are attached to the rest of the DNA using DNA ligase, which creates a phosphodiester bond.

Most mistakes are corrected, and if they are not, they may result in a mutation defined as a permanent change in the DNA sequence. Mutations can be of many types, such as substitution, deletion, insertion, and translocation. Mutations in repair genes may lead to serious consequences such as cancer. Mutations can be induced or may occur spontaneously.


DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 10,000 to 1,000,000 molecular lesions per cell per day. [2] While this constitutes only 0.000165% of the human genome's approximately 6 billion bases, unrepaired lesions in critical genes (such as tumor suppressor genes) can impede a cell's ability to carry out its function and appreciably increase the likelihood of tumor formation and contribute to tumour heterogeneity.

The vast majority of DNA damage affects the primary structure of the double helix that is, the bases themselves are chemically modified. These modifications can in turn disrupt the molecules' regular helical structure by introducing non-native chemical bonds or bulky adducts that do not fit in the standard double helix. Unlike proteins and RNA, DNA usually lacks tertiary structure and therefore damage or disturbance does not occur at that level. DNA is, however, supercoiled and wound around "packaging" proteins called histones (in eukaryotes), and both superstructures are vulnerable to the effects of DNA damage.

Sources Edit

DNA damage can be subdivided into two main types:

    damage such as attack by reactive oxygen species produced from normal metabolic byproducts (spontaneous mutation), especially the process of oxidative deamination
    1. also includes replication errors
    1. ultraviolet [UV 200–400 nm] radiation from the sun or other artificial light sources
    2. other radiation frequencies, including x-rays and gamma rays or thermal disruption
    3. certain planttoxins
    4. human-made mutagenic chemicals, especially aromatic compounds that act as DNA intercalating agents[8]

    The replication of damaged DNA before cell division can lead to the incorporation of wrong bases opposite damaged ones. Daughter cells that inherit these wrong bases carry mutations from which the original DNA sequence is unrecoverable (except in the rare case of a back mutation, for example, through gene conversion).

    Types Edit

    There are several types of damage to DNA due to endogenous cellular processes:

    1. oxidation of bases [e.g. 8-oxo-7,8-dihydroguanine (8-oxoG)] and generation of DNA strand interruptions from reactive oxygen species,
    2. alkylation of bases (usually methylation), such as formation of 7-methylguanosine, 1-methyladenine, 6-O-Methylguanine
    3. hydrolysis of bases, such as deamination, depurination, and depyrimidination. (e.g., benzo[a]pyrene diol epoxide-dG adduct, aristolactam I-dA adduct)
    4. mismatch of bases, due to errors in DNA replication, in which the wrong DNA base is stitched into place in a newly forming DNA strand, or a DNA base is skipped over or mistakenly inserted.
    5. Monoadduct damage cause by change in single nitrogenous base of DNA
    6. Diadduct damage

    Damage caused by exogenous agents comes in many forms. Some examples are:

    1. UV-B light causes crosslinking between adjacent cytosine and thymine bases creating pyrimidine dimers. This is called direct DNA damage.
    2. UV-A light creates mostly free radicals. The damage caused by free radicals is called indirect DNA damage.
    3. Ionizing radiation such as that created by radioactive decay or in cosmic rays causes breaks in DNA strands. Intermediate-level ionizing radiation may induce irreparable DNA damage (leading to replicational and transcriptional errors needed for neoplasia or may trigger viral interactions) leading to pre-mature aging and cancer.
    4. Thermal disruption at elevated temperature increases the rate of depurination (loss of purine bases from the DNA backbone) and single-strand breaks. For example, hydrolytic depurination is seen in the thermophilic bacteria, which grow in hot springs at 40–80 °C. [9][10] The rate of depurination (300 purine residues per genome per generation) is too high in these species to be repaired by normal repair machinery, hence a possibility of an adaptive response cannot be ruled out.
    5. Industrial chemicals such as vinyl chloride and hydrogen peroxide, and environmental chemicals such as polycyclic aromatic hydrocarbons found in smoke, soot and tar create a huge diversity of DNA adducts- ethenobases, oxidized bases, alkylated phosphotriesters and crosslinking of DNA, just to name a few.

    UV damage, alkylation/methylation, X-ray damage and oxidative damage are examples of induced damage. Spontaneous damage can include the loss of a base, deamination, sugar ring puckering and tautomeric shift. Constitutive (spontaneous) DNA damage caused by endogenous oxidants can be detected as a low level of histone H2AX phosphorylation in untreated cells. [11]

    Nuclear versus mitochondrial Edit

    In human cells, and eukaryotic cells in general, DNA is found in two cellular locations – inside the nucleus and inside the mitochondria. Nuclear DNA (nDNA) exists as chromatin during non-replicative stages of the cell cycle and is condensed into aggregate structures known as chromosomes during cell division. In either state the DNA is highly compacted and wound up around bead-like proteins called histones. Whenever a cell needs to express the genetic information encoded in its nDNA the required chromosomal region is unravelled, genes located therein are expressed, and then the region is condensed back to its resting conformation. Mitochondrial DNA (mtDNA) is located inside mitochondria organelles, exists in multiple copies, and is also tightly associated with a number of proteins to form a complex known as the nucleoid. Inside mitochondria, reactive oxygen species (ROS), or free radicals, byproducts of the constant production of adenosine triphosphate (ATP) via oxidative phosphorylation, create a highly oxidative environment that is known to damage mtDNA. A critical enzyme in counteracting the toxicity of these species is superoxide dismutase, which is present in both the mitochondria and cytoplasm of eukaryotic cells.

    Senescence and apoptosis Edit

    Senescence, an irreversible process in which the cell no longer divides, is a protective response to the shortening of the chromosome ends. The telomeres are long regions of repetitive noncoding DNA that cap chromosomes and undergo partial degradation each time a cell undergoes division (see Hayflick limit). [12] In contrast, quiescence is a reversible state of cellular dormancy that is unrelated to genome damage (see cell cycle). Senescence in cells may serve as a functional alternative to apoptosis in cases where the physical presence of a cell for spatial reasons is required by the organism, [13] which serves as a "last resort" mechanism to prevent a cell with damaged DNA from replicating inappropriately in the absence of pro-growth cellular signaling. Unregulated cell division can lead to the formation of a tumor (see cancer), which is potentially lethal to an organism. Therefore, the induction of senescence and apoptosis is considered to be part of a strategy of protection against cancer. [14]

    Mutation Edit

    It is important to distinguish between DNA damage and mutation, the two major types of error in DNA. DNA damage and mutation are fundamentally different. Damage results in physical abnormalities in the DNA, such as single- and double-strand breaks, 8-hydroxydeoxyguanosine residues, and polycyclic aromatic hydrocarbon adducts. DNA damage can be recognized by enzymes, and thus can be correctly repaired if redundant information, such as the undamaged sequence in the complementary DNA strand or in a homologous chromosome, is available for copying. If a cell retains DNA damage, transcription of a gene can be prevented, and thus translation into a protein will also be blocked. Replication may also be blocked or the cell may die.

    In contrast to DNA damage, a mutation is a change in the base sequence of the DNA. A mutation cannot be recognized by enzymes once the base change is present in both DNA strands, and thus a mutation cannot be repaired. At the cellular level, mutations can cause alterations in protein function and regulation. Mutations are replicated when the cell replicates. In a population of cells, mutant cells will increase or decrease in frequency according to the effects of the mutation on the ability of the cell to survive and reproduce.

    Although distinctly different from each other, DNA damage and mutation are related because DNA damage often causes errors of DNA synthesis during replication or repair these errors are a major source of mutation.

    Given these properties of DNA damage and mutation, it can be seen that DNA damage is a special problem in non-dividing or slowly-dividing cells, where unrepaired damage will tend to accumulate over time. On the other hand, in rapidly-dividing cells, unrepaired DNA damage that does not kill the cell by blocking replication will tend to cause replication errors and thus mutation. The great majority of mutations that are not neutral in their effect are deleterious to a cell's survival. Thus, in a population of cells composing a tissue with replicating cells, mutant cells will tend to be lost. However, infrequent mutations that provide a survival advantage will tend to clonally expand at the expense of neighboring cells in the tissue. This advantage to the cell is disadvantageous to the whole organism, because such mutant cells can give rise to cancer. Thus, DNA damage in frequently dividing cells, because it gives rise to mutations, is a prominent cause of cancer. In contrast, DNA damage in infrequently-dividing cells is likely a prominent cause of aging. [15]

    Cells cannot function if DNA damage corrupts the integrity and accessibility of essential information in the genome (but cells remain superficially functional when non-essential genes are missing or damaged). Depending on the type of damage inflicted on the DNA's double helical structure, a variety of repair strategies have evolved to restore lost information. If possible, cells use the unmodified complementary strand of the DNA or the sister chromatid as a template to recover the original information. Without access to a template, cells use an error-prone recovery mechanism known as translesion synthesis as a last resort.

    Damage to DNA alters the spatial configuration of the helix, and such alterations can be detected by the cell. Once damage is localized, specific DNA repair molecules bind at or near the site of damage, inducing other molecules to bind and form a complex that enables the actual repair to take place.

    Direct reversal Edit

    Cells are known to eliminate three types of damage to their DNA by chemically reversing it. These mechanisms do not require a template, since the types of damage they counteract can occur in only one of the four bases. Such direct reversal mechanisms are specific to the type of damage incurred and do not involve breakage of the phosphodiester backbone. The formation of pyrimidine dimers upon irradiation with UV light results in an abnormal covalent bond between adjacent pyrimidine bases. The photoreactivation process directly reverses this damage by the action of the enzyme photolyase, whose activation is obligately dependent on energy absorbed from blue/UV light (300–500 nm wavelength) to promote catalysis. [16] Photolyase, an old enzyme present in bacteria, fungi, and most animals no longer functions in humans, [17] who instead use nucleotide excision repair to repair damage from UV irradiation. Another type of damage, methylation of guanine bases, is directly reversed by the protein methyl guanine methyl transferase (MGMT), the bacterial equivalent of which is called ogt. This is an expensive process because each MGMT molecule can be used only once that is, the reaction is stoichiometric rather than catalytic. [18] A generalized response to methylating agents in bacteria is known as the adaptive response and confers a level of resistance to alkylating agents upon sustained exposure by upregulation of alkylation repair enzymes. [19] The third type of DNA damage reversed by cells is certain methylation of the bases cytosine and adenine.

    Single-strand damage Edit

    When only one of the two strands of a double helix has a defect, the other strand can be used as a template to guide the correction of the damaged strand. In order to repair damage to one of the two paired molecules of DNA, there exist a number of excision repair mechanisms that remove the damaged nucleotide and replace it with an undamaged nucleotide complementary to that found in the undamaged DNA strand. [18]

      (BER): damaged single bases or nucleotides are most commonly repaired by removing the base or the nucleotide involved and then inserting the correct base or nucleotide. In base excision repair, a glycosylase[20] enzyme removes the damaged base from the DNA by cleaving the bond between the base and the deoxyribose. These enzymes remove a single base to create an apurinic or apyrimidinic site (AP site). [20] Enzymes called AP endonucleasesnick the damaged DNA backbone at the AP site. DNA polymerase then removes the damaged region using its 5’ to 3’ exonuclease activity and correctly synthesizes the new strand using the complementary strand as a template. [20] The gap is then sealed by enzyme DNA ligase. [21] (NER): bulky, helix-distorting damage, such as pyrimidine dimerization caused by UV light is usually repaired by a three-step process. First the damage is recognized, then 12-24 nucleotide-long strands of DNA are removed both upstream and downstream of the damage site by endonucleases, and the removed DNA region is then resynthesized. [22] NER is a highly evolutionarily conserved repair mechanism and is used in nearly all eukaryotic and prokaryotic cells. [22] In prokaryotes, NER is mediated by Uvr proteins. [22] In eukaryotes, many more proteins are involved, although the general strategy is the same. [22] systems are present in essentially all cells to correct errors that are not corrected by proofreading. These systems consist of at least two proteins. One detects the mismatch, and the other recruits an endonuclease that cleaves the newly synthesized DNA strand close to the region of damage. In E. coli , the proteins involved are the Mut class proteins: MutS, MutL, and MutH. In most Eukaryotes, the analog for MutS is MSH and the analog for MutL is MLH. MutH is only present in bacteria. This is followed by removal of damaged region by an exonuclease, resynthesis by DNA polymerase, and nick sealing by DNA ligase. [23]

    Double-strand breaks Edit

    Double-strand breaks, in which both strands in the double helix are severed, are particularly hazardous to the cell because they can lead to genome rearrangements. In fact, when a double-strand break is accompanied by a cross-linkage joining the two strands at the same point, neither strand can be used as a template for the repair mechanisms, so that the cell will not be able to complete mitosis when it next divides, and will either die or, in rare cases, undergo a mutation. [3] [4] Three mechanisms exist to repair double-strand breaks (DSBs): non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination (HR). [18] [24] In an in vitro system, MMEJ occurred in mammalian cells at the levels of 10–20% of HR when both HR and NHEJ mechanisms were also available. [25]

    In NHEJ, DNA Ligase IV, a specialized DNA ligase that forms a complex with the cofactor XRCC4, directly joins the two ends. [26] To guide accurate repair, NHEJ relies on short homologous sequences called microhomologies present on the single-stranded tails of the DNA ends to be joined. If these overhangs are compatible, repair is usually accurate. [27] [28] [29] [30] NHEJ can also introduce mutations during repair. Loss of damaged nucleotides at the break site can lead to deletions, and joining of nonmatching termini forms insertions or translocations. NHEJ is especially important before the cell has replicated its DNA, since there is no template available for repair by homologous recombination. There are "backup" NHEJ pathways in higher eukaryotes. [31] Besides its role as a genome caretaker, NHEJ is required for joining hairpin-capped double-strand breaks induced during V(D)J recombination, the process that generates diversity in B-cell and T-cell receptors in the vertebrate immune system. [32]

    Homologous recombination requires the presence of an identical or nearly identical sequence to be used as a template for repair of the break. The enzymatic machinery responsible for this repair process is nearly identical to the machinery responsible for chromosomal crossover during meiosis. This pathway allows a damaged chromosome to be repaired using a sister chromatid (available in G2 after DNA replication) or a homologous chromosome as a template. DSBs caused by the replication machinery attempting to synthesize across a single-strand break or unrepaired lesion cause collapse of the replication fork and are typically repaired by recombination.

    MMEJ starts with short-range end resection by MRE11 nuclease on either side of a double-strand break to reveal microhomology regions. [25] In further steps, [33] Poly (ADP-ribose) polymerase 1 (PARP1) is required and may be an early step in MMEJ. There is pairing of microhomology regions followed by recruitment of flap structure-specific endonuclease 1 (FEN1) to remove overhanging flaps. This is followed by recruitment of XRCC1–LIG3 to the site for ligating the DNA ends, leading to an intact DNA. MMEJ is always accompanied by a deletion, so that MMEJ is a mutagenic pathway for DNA repair. [34]

    The extremophile Deinococcus radiodurans has a remarkable ability to survive DNA damage from ionizing radiation and other sources. At least two copies of the genome, with random DNA breaks, can form DNA fragments through annealing. Partially overlapping fragments are then used for synthesis of homologous regions through a moving D-loop that can continue extension until they find complementary partner strands. In the final step there is crossover by means of RecA-dependent homologous recombination. [35]

    Topoisomerases introduce both single- and double-strand breaks in the course of changing the DNA's state of supercoiling, which is especially common in regions near an open replication fork. Such breaks are not considered DNA damage because they are a natural intermediate in the topoisomerase biochemical mechanism and are immediately repaired by the enzymes that created them.

    Translesion synthesis Edit

    Translesion synthesis (TLS) is a DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sites. [36] It involves switching out regular DNA polymerases for specialized translesion polymerases (i.e. DNA polymerase IV or V, from the Y Polymerase family), often with larger active sites that can facilitate the insertion of bases opposite damaged nucleotides. The polymerase switching is thought to be mediated by, among other factors, the post-translational modification of the replication processivity factor PCNA. Translesion synthesis polymerases often have low fidelity (high propensity to insert wrong bases) on undamaged templates relative to regular polymerases. However, many are extremely efficient at inserting correct bases opposite specific types of damage. For example, Pol η mediates error-free bypass of lesions induced by UV irradiation, whereas Pol ι introduces mutations at these sites. Pol η is known to add the first adenine across the T^T photodimer using Watson-Crick base pairing and the second adenine will be added in its syn conformation using Hoogsteen base pairing. From a cellular perspective, risking the introduction of point mutations during translesion synthesis may be preferable to resorting to more drastic mechanisms of DNA repair, which may cause gross chromosomal aberrations or cell death. In short, the process involves specialized polymerases either bypassing or repairing lesions at locations of stalled DNA replication. For example, Human DNA polymerase eta can bypass complex DNA lesions like guanine-thymine intra-strand crosslink, G[8,5-Me]T, although it can cause targeted and semi-targeted mutations. [37] Paromita Raychaudhury and Ashis Basu [38] studied the toxicity and mutagenesis of the same lesion in Escherichia coli by replicating a G[8,5-Me]T-modified plasmid in E. coli with specific DNA polymerase knockouts. Viability was very low in a strain lacking pol II, pol IV, and pol V, the three SOS-inducible DNA polymerases, indicating that translesion synthesis is conducted primarily by these specialized DNA polymerases. A bypass platform is provided to these polymerases by Proliferating cell nuclear antigen (PCNA). Under normal circumstances, PCNA bound to polymerases replicates the DNA. At a site of lesion, PCNA is ubiquitinated, or modified, by the RAD6/RAD18 proteins to provide a platform for the specialized polymerases to bypass the lesion and resume DNA replication. [39] [40] After translesion synthesis, extension is required. This extension can be carried out by a replicative polymerase if the TLS is error-free, as in the case of Pol η, yet if TLS results in a mismatch, a specialized polymerase is needed to extend it Pol ζ. Pol ζ is unique in that it can extend terminal mismatches, whereas more processive polymerases cannot. So when a lesion is encountered, the replication fork will stall, PCNA will switch from a processive polymerase to a TLS polymerase such as Pol ι to fix the lesion, then PCNA may switch to Pol ζ to extend the mismatch, and last PCNA will switch to the processive polymerase to continue replication.

    Cells exposed to ionizing radiation, ultraviolet light or chemicals are prone to acquire multiple sites of bulky DNA lesions and double-strand breaks. Moreover, DNA damaging agents can damage other biomolecules such as proteins, carbohydrates, lipids, and RNA. The accumulation of damage, to be specific, double-strand breaks or adducts stalling the replication forks, are among known stimulation signals for a global response to DNA damage. [41] The global response to damage is an act directed toward the cells' own preservation and triggers multiple pathways of macromolecular repair, lesion bypass, tolerance, or apoptosis. The common features of global response are induction of multiple genes, cell cycle arrest, and inhibition of cell division.

    Initial steps Edit

    The packaging of eukaryotic DNA into chromatin presents a barrier to all DNA-based processes that require recruitment of enzymes to their sites of action. To allow DNA repair, the chromatin must be remodeled. In eukaryotes, ATP dependent chromatin remodeling complexes and histone-modifying enzymes are two predominant factors employed to accomplish this remodeling process. [42]

    Chromatin relaxation occurs rapidly at the site of a DNA damage. [43] [44] In one of the earliest steps, the stress-activated protein kinase, c-Jun N-terminal kinase (JNK), phosphorylates SIRT6 on serine 10 in response to double-strand breaks or other DNA damage. [45] This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites, and is required for efficient recruitment of poly (ADP-ribose) polymerase 1 (PARP1) to DNA break sites and for efficient repair of DSBs. [45] PARP1 protein starts to appear at DNA damage sites in less than a second, with half maximum accumulation within 1.6 seconds after the damage occurs. [46] PARP1 synthesizes polymeric adenosine diphosphate ribose (poly (ADP-ribose) or PAR) chains on itself. Next the chromatin remodeler ALC1 quickly attaches to the product of PARP1 action, a poly-ADP ribose chain, and ALC1 completes arrival at the DNA damage within 10 seconds of the occurrence of the damage. [44] About half of the maximum chromatin relaxation, presumably due to action of ALC1, occurs by 10 seconds. [44] This then allows recruitment of the DNA repair enzyme MRE11, to initiate DNA repair, within 13 seconds. [46]

    γH2AX, the phosphorylated form of H2AX is also involved in the early steps leading to chromatin decondensation after DNA double-strand breaks. The histone variant H2AX constitutes about 10% of the H2A histones in human chromatin. [47] γH2AX (H2AX phosphorylated on serine 139) can be detected as soon as 20 seconds after irradiation of cells (with DNA double-strand break formation), and half maximum accumulation of γH2AX occurs in one minute. [47] The extent of chromatin with phosphorylated γH2AX is about two million base pairs at the site of a DNA double-strand break. [47] γH2AX does not, itself, cause chromatin decondensation, but within 30 seconds of irradiation, RNF8 protein can be detected in association with γH2AX. [48] RNF8 mediates extensive chromatin decondensation, through its subsequent interaction with CHD4, [49] a component of the nucleosome remodeling and deacetylase complex NuRD.

    DDB2 occurs in a heterodimeric complex with DDB1. This complex further complexes with the ubiquitin ligase protein CUL4A [50] and with PARP1. [51] This larger complex rapidly associates with UV-induced damage within chromatin, with half-maximum association completed in 40 seconds. [50] The PARP1 protein, attached to both DDB1 and DDB2, then PARylates (creates a poly-ADP ribose chain) on DDB2 that attracts the DNA remodeling protein ALC1. [51] Action of ALC1 relaxes the chromatin at the site of UV damage to DNA. This relaxation allows other proteins in the nucleotide excision repair pathway to enter the chromatin and repair UV-induced cyclobutane pyrimidine dimer damages.

    After rapid chromatin remodeling, cell cycle checkpoints are activated to allow DNA repair to occur before the cell cycle progresses. First, two kinases, ATM and ATR are activated within 5 or 6 minutes after DNA is damaged. This is followed by phosphorylation of the cell cycle checkpoint protein Chk1, initiating its function, about 10 minutes after DNA is damaged. [52]

    DNA damage checkpoints Edit

    After DNA damage, cell cycle checkpoints are activated. Checkpoint activation pauses the cell cycle and gives the cell time to repair the damage before continuing to divide. DNA damage checkpoints occur at the G1/S and G2/M boundaries. An intra-S checkpoint also exists. Checkpoint activation is controlled by two master kinases, ATM and ATR. ATM responds to DNA double-strand breaks and disruptions in chromatin structure, [53] whereas ATR primarily responds to stalled replication forks. These kinases phosphorylate downstream targets in a signal transduction cascade, eventually leading to cell cycle arrest. A class of checkpoint mediator proteins including BRCA1, MDC1, and 53BP1 has also been identified. [54] These proteins seem to be required for transmitting the checkpoint activation signal to downstream proteins.

    DNA damage checkpoint is a signal transduction pathway that blocks cell cycle progression in G1, G2 and metaphase and slows down the rate of S phase progression when DNA is damaged. It leads to a pause in cell cycle allowing the cell time to repair the damage before continuing to divide.

    Checkpoint Proteins can be separated into four groups: phosphatidylinositol 3-kinase (PI3K)-like protein kinase, proliferating cell nuclear antigen (PCNA)-like group, two serine/threonine(S/T) kinases and their adaptors. Central to all DNA damage induced checkpoints responses is a pair of large protein kinases belonging to the first group of PI3K-like protein kinases-the ATM (Ataxia telangiectasia mutated) and ATR (Ataxia- and Rad-related) kinases, whose sequence and functions have been well conserved in evolution. All DNA damage response requires either ATM or ATR because they have the ability to bind to the chromosomes at the site of DNA damage, together with accessory proteins that are platforms on which DNA damage response components and DNA repair complexes can be assembled.

    An important downstream target of ATM and ATR is p53, as it is required for inducing apoptosis following DNA damage. [55] The cyclin-dependent kinase inhibitor p21 is induced by both p53-dependent and p53-independent mechanisms and can arrest the cell cycle at the G1/S and G2/M checkpoints by deactivating cyclin/cyclin-dependent kinase complexes. [56]

    The prokaryotic SOS response Edit

    The SOS response is the changes in gene expression in Escherichia coli and other bacteria in response to extensive DNA damage. The prokaryotic SOS system is regulated by two key proteins: LexA and RecA. The LexA homodimer is a transcriptional repressor that binds to operator sequences commonly referred to as SOS boxes. In Escherichia coli it is known that LexA regulates transcription of approximately 48 genes including the lexA and recA genes. [57] The SOS response is known to be widespread in the Bacteria domain, but it is mostly absent in some bacterial phyla, like the Spirochetes. [58] The most common cellular signals activating the SOS response are regions of single-stranded DNA (ssDNA), arising from stalled replication forks or double-strand breaks, which are processed by DNA helicase to separate the two DNA strands. [41] In the initiation step, RecA protein binds to ssDNA in an ATP hydrolysis driven reaction creating RecA–ssDNA filaments. RecA–ssDNA filaments activate LexA autoprotease activity, which ultimately leads to cleavage of LexA dimer and subsequent LexA degradation. The loss of LexA repressor induces transcription of the SOS genes and allows for further signal induction, inhibition of cell division and an increase in levels of proteins responsible for damage processing.

    In Escherichia coli, SOS boxes are 20-nucleotide long sequences near promoters with palindromic structure and a high degree of sequence conservation. In other classes and phyla, the sequence of SOS boxes varies considerably, with different length and composition, but it is always highly conserved and one of the strongest short signals in the genome. [58] The high information content of SOS boxes permits differential binding of LexA to different promoters and allows for timing of the SOS response. The lesion repair genes are induced at the beginning of SOS response. The error-prone translesion polymerases, for example, UmuCD'2 (also called DNA polymerase V), are induced later on as a last resort. [59] Once the DNA damage is repaired or bypassed using polymerases or through recombination, the amount of single-stranded DNA in cells is decreased, lowering the amounts of RecA filaments decreases cleavage activity of LexA homodimer, which then binds to the SOS boxes near promoters and restores normal gene expression.

    Eukaryotic transcriptional responses to DNA damage Edit

    Eukaryotic cells exposed to DNA damaging agents also activate important defensive pathways by inducing multiple proteins involved in DNA repair, cell cycle checkpoint control, protein trafficking and degradation. Such genome wide transcriptional response is very complex and tightly regulated, thus allowing coordinated global response to damage. Exposure of yeast Saccharomyces cerevisiae to DNA damaging agents results in overlapping but distinct transcriptional profiles. Similarities to environmental shock response indicates that a general global stress response pathway exist at the level of transcriptional activation. In contrast, different human cell types respond to damage differently indicating an absence of a common global response. The probable explanation for this difference between yeast and human cells may be in the heterogeneity of mammalian cells. In an animal different types of cells are distributed among different organs that have evolved different sensitivities to DNA damage. [60]

    In general global response to DNA damage involves expression of multiple genes responsible for postreplication repair, homologous recombination, nucleotide excision repair, DNA damage checkpoint, global transcriptional activation, genes controlling mRNA decay, and many others. A large amount of damage to a cell leaves it with an important decision: undergo apoptosis and die, or survive at the cost of living with a modified genome. An increase in tolerance to damage can lead to an increased rate of survival that will allow a greater accumulation of mutations. Yeast Rev1 and human polymerase η are members of [Y family translesion DNA polymerases present during global response to DNA damage and are responsible for enhanced mutagenesis during a global response to DNA damage in eukaryotes. [41]

    Pathological effects of poor DNA repair Edit

    Experimental animals with genetic deficiencies in DNA repair often show decreased life span and increased cancer incidence. [15] For example, mice deficient in the dominant NHEJ pathway and in telomere maintenance mechanisms get lymphoma and infections more often, and, as a consequence, have shorter lifespans than wild-type mice. [61] In similar manner, mice deficient in a key repair and transcription protein that unwinds DNA helices have premature onset of aging-related diseases and consequent shortening of lifespan. [62] However, not every DNA repair deficiency creates exactly the predicted effects mice deficient in the NER pathway exhibited shortened life span without correspondingly higher rates of mutation. [63]

    If the rate of DNA damage exceeds the capacity of the cell to repair it, the accumulation of errors can overwhelm the cell and result in early senescence, apoptosis, or cancer. Inherited diseases associated with faulty DNA repair functioning result in premature aging, [15] increased sensitivity to carcinogens, and correspondingly increased cancer risk (see below). On the other hand, organisms with enhanced DNA repair systems, such as Deinococcus radiodurans, the most radiation-resistant known organism, exhibit remarkable resistance to the double-strand break-inducing effects of radioactivity, likely due to enhanced efficiency of DNA repair and especially NHEJ. [64]

    Longevity and caloric restriction Edit

    A number of individual genes have been identified as influencing variations in life span within a population of organisms. The effects of these genes is strongly dependent on the environment, in particular, on the organism's diet. Caloric restriction reproducibly results in extended lifespan in a variety of organisms, likely via nutrient sensing pathways and decreased metabolic rate. The molecular mechanisms by which such restriction results in lengthened lifespan are as yet unclear (see [65] for some discussion) however, the behavior of many genes known to be involved in DNA repair is altered under conditions of caloric restriction. Several agents reported to have anti-aging properties have been shown to attenuate constitutive level of mTOR signaling, an evidence of reduction of metabolic activity, and concurrently to reduce constitutive level of DNA damage induced by endogenously generated reactive oxygen species. [66]

    For example, increasing the gene dosage of the gene SIR-2, which regulates DNA packaging in the nematode worm Caenorhabditis elegans, can significantly extend lifespan. [67] The mammalian homolog of SIR-2 is known to induce downstream DNA repair factors involved in NHEJ, an activity that is especially promoted under conditions of caloric restriction. [68] Caloric restriction has been closely linked to the rate of base excision repair in the nuclear DNA of rodents, [69] although similar effects have not been observed in mitochondrial DNA. [70]

    The C. elegans gene AGE-1, an upstream effector of DNA repair pathways, confers dramatically extended life span under free-feeding conditions but leads to a decrease in reproductive fitness under conditions of caloric restriction. [71] This observation supports the pleiotropy theory of the biological origins of aging, which suggests that genes conferring a large survival advantage early in life will be selected for even if they carry a corresponding disadvantage late in life.

    Hereditary DNA repair disorders Edit

    Defects in the NER mechanism are responsible for several genetic disorders, including:

      : hypersensitivity to sunlight/UV, resulting in increased skin cancer incidence and premature aging : hypersensitivity to UV and chemical agents : sensitive skin, brittle hair and nails

    Mental retardation often accompanies the latter two disorders, suggesting increased vulnerability of developmental neurons.

    Other DNA repair disorders include:

      : premature aging and retarded growth : sunlight hypersensitivity, high incidence of malignancies (especially leukemias). : sensitivity to ionizing radiation and some chemical agents

    All of the above diseases are often called "segmental progerias" ("accelerated aging diseases") because their victims appear elderly and suffer from aging-related diseases at an abnormally young age, while not manifesting all the symptoms of old age.

    Other diseases associated with reduced DNA repair function include Fanconi anemia, hereditary breast cancer and hereditary colon cancer.

    Because of inherent limitations in the DNA repair mechanisms, if humans lived long enough, they would all eventually develop cancer. [72] [73] There are at least 34 Inherited human DNA repair gene mutations that increase cancer risk. Many of these mutations cause DNA repair to be less effective than normal. In particular, Hereditary nonpolyposis colorectal cancer (HNPCC) is strongly associated with specific mutations in the DNA mismatch repair pathway. BRCA1 and BRCA2, two important genes whose mutations confer a hugely increased risk of breast cancer on carriers, [74] are both associated with a large number of DNA repair pathways, especially NHEJ and homologous recombination.

    Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage, resulting in cell death. Cells that are most rapidly dividing – most typically cancer cells – are preferentially affected. The side-effect is that other non-cancerous but rapidly dividing cells such as progenitor cells in the gut, skin, and hematopoietic system are also affected. Modern cancer treatments attempt to localize the DNA damage to cells and tissues only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body). In the context of therapies targeting DNA damage response genes, the latter approach has been termed 'synthetic lethality'. [75]

    Perhaps the most well-known of these 'synthetic lethality' drugs is the poly(ADP-ribose) polymerase 1 (PARP1) inhibitor olaparib, which was approved by the Food and Drug Administration in 2015 for the treatment in women of BRCA-defective ovarian cancer. Tumor cells with partial loss of DNA damage response (specifically, homologous recombination repair) are dependent on another mechanism – single-strand break repair – which is a mechanism consisting, in part, of the PARP1 gene product. [76] Olaparib is combined with chemotherapeutics to inhibit single-strand break repair induced by DNA damage caused by the co-administered chemotherapy. Tumor cells relying on this residual DNA repair mechanism are unable to repair the damage and hence are not able to survive and proliferate, whereas normal cells can repair the damage with the functioning homologous recombination mechanism.

    Many other drugs for use against other residual DNA repair mechanisms commonly found in cancer are currently under investigation. However, synthetic lethality therapeutic approaches have been questioned due to emerging evidence of acquired resistance, achieved through rewiring of DNA damage response pathways and reversion of previously-inhibited defects. [77]

    DNA repair defects in cancer Edit

    It has become apparent over the past several years that the DNA damage response acts as a barrier to the malignant transformation of preneoplastic cells. [78] Previous studies have shown an elevated DNA damage response in cell-culture models with oncogene activation [79] and preneoplastic colon adenomas. [80] DNA damage response mechanisms trigger cell-cycle arrest, and attempt to repair DNA lesions or promote cell death/senescence if repair is not possible. Replication stress is observed in preneoplastic cells due to increased proliferation signals from oncogenic mutations. Replication stress is characterized by: increased replication initiation/origin firing increased transcription and collisions of transcription-replication complexes nucleotide deficiency increase in reactive oxygen species (ROS). [81]

    Replication stress, along with the selection for inactivating mutations in DNA damage response genes in the evolution of the tumor, [82] leads to downregulation and/or loss of some DNA damage response mechanisms, and hence loss of DNA repair and/or senescence/programmed cell death. In experimental mouse models, loss of DNA damage response-mediated cell senescence was observed after using a short hairpin RNA (shRNA) to inhibit the double-strand break response kinase ataxia telangiectasia (ATM), leading to increased tumor size and invasiveness. [80] Humans born with inherited defects in DNA repair mechanisms (for example, Li-Fraumeni syndrome) have a higher cancer risk. [83]

    The prevalence of DNA damage response mutations differs across cancer types for example, 30% of breast invasive carcinomas have mutations in genes involved in homologous recombination. [78] In cancer, downregulation is observed across all DNA damage response mechanisms (base excision repair (BER), nucleotide excision repair (NER), DNA mismatch repair (MMR), homologous recombination repair (HR), non-homologous end joining (NHEJ) and translesion DNA synthesis (TLS). [84] As well as mutations to DNA damage repair genes, mutations also arise in the genes responsible for arresting the cell cycle to allow sufficient time for DNA repair to occur, and some genes are involved in both DNA damage repair and cell cycle checkpoint control, for example ATM and checkpoint kinase 2 (CHEK2) – a tumor suppressor that is often absent or downregulated in non-small cell lung cancer. [85]

    ATM x x x
    ATR x x x
    PAXIP x x
    RPA x x x
    BRCA1 x x
    BRCA2 x x
    RAD51 x x
    RFC x x x
    XRCC1 x x
    PCNA x x x
    PARP1 x x
    ERCC1 x x x x
    MSH3 x x x

    Table: Genes involved in DNA damage response pathways and frequently mutated in cancer (HR = homologous recombination NHEJ = non-homologous end joining SSA = single-strand annealing FA = fanconi anemia pathway BER = base excision repair NER = nucleotide excision repair MMR = mismatch repair)

    Epigenetic DNA repair defects in cancer Edit

    Classically, cancer has been viewed as a set of diseases that are driven by progressive genetic abnormalities that include mutations in tumour-suppressor genes and oncogenes, and chromosomal aberrations. However, it has become apparent that cancer is also driven by epigenetic alterations. [86]

    Epigenetic alterations refer to functionally relevant modifications to the genome that do not involve a change in the nucleotide sequence. Examples of such modifications are changes in DNA methylation (hypermethylation and hypomethylation) and histone modification, [87] changes in chromosomal architecture (caused by inappropriate expression of proteins such as HMGA2 or HMGA1) [88] and changes caused by microRNAs. Each of these epigenetic alterations serves to regulate gene expression without altering the underlying DNA sequence. These changes usually remain through cell divisions, last for multiple cell generations, and can be considered to be epimutations (equivalent to mutations).

    While large numbers of epigenetic alterations are found in cancers, the epigenetic alterations in DNA repair genes, causing reduced expression of DNA repair proteins, appear to be particularly important. Such alterations are thought to occur early in progression to cancer and to be a likely cause of the genetic instability characteristic of cancers. [89] [90] [91]

    Reduced expression of DNA repair genes causes deficient DNA repair. When DNA repair is deficient DNA damages remain in cells at a higher than usual level and these excess damages cause increased frequencies of mutation or epimutation. Mutation rates increase substantially in cells defective in DNA mismatch repair [92] [93] or in homologous recombinational repair (HRR). [94] Chromosomal rearrangements and aneuploidy also increase in HRR defective cells. [95]

    Higher levels of DNA damage not only cause increased mutation, but also cause increased epimutation. During repair of DNA double strand breaks, or repair of other DNA damages, incompletely cleared sites of repair can cause epigenetic gene silencing. [96] [97]

    Deficient expression of DNA repair proteins due to an inherited mutation can cause increased risk of cancer. Individuals with an inherited impairment in any of 34 DNA repair genes (see article DNA repair-deficiency disorder) have an increased risk of cancer, with some defects causing up to a 100% lifetime chance of cancer (e.g. p53 mutations). [98] However, such germline mutations (which cause highly penetrant cancer syndromes) are the cause of only about 1 percent of cancers. [99]

    Frequencies of epimutations in DNA repair genes Edit

    Deficiencies in DNA repair enzymes are occasionally caused by a newly arising somatic mutation in a DNA repair gene, but are much more frequently caused by epigenetic alterations that reduce or silence expression of DNA repair genes. For example, when 113 colorectal cancers were examined in sequence, only four had a missense mutation in the DNA repair gene MGMT, while the majority had reduced MGMT expression due to methylation of the MGMT promoter region (an epigenetic alteration). [100] Five different studies found that between 40% and 90% of colorectal cancers have reduced MGMT expression due to methylation of the MGMT promoter region. [101] [102] [103] [104] [105]

    Similarly, out of 119 cases of mismatch repair-deficient colorectal cancers that lacked DNA repair gene PMS2 expression, PMS2 was deficient in 6 due to mutations in the PMS2 gene, while in 103 cases PMS2 expression was deficient because its pairing partner MLH1 was repressed due to promoter methylation (PMS2 protein is unstable in the absence of MLH1). [106] In the other 10 cases, loss of PMS2 expression was likely due to epigenetic overexpression of the microRNA, miR-155, which down-regulates MLH1. [107]

    In a further example, epigenetic defects were found in various cancers (e.g. breast, ovarian, colorectal and head and neck). Two or three deficiencies in the expression of ERCC1, XPF or PMS2 occur simultaneously in the majority of 49 colon cancers evaluated by Facista et al. [108]

    The chart in this section shows some frequent DNA damaging agents, examples of DNA lesions they cause, and the pathways that deal with these DNA damages. At least 169 enzymes are either directly employed in DNA repair or influence DNA repair processes. [109] Of these, 83 are directly employed in repairing the 5 types of DNA damages illustrated in the chart.

    Some of the more well studied genes central to these repair processes are shown in the chart. The gene designations shown in red, gray or cyan indicate genes frequently epigenetically altered in various types of cancers. Wikipedia articles on each of the genes highlighted by red, gray or cyan describe the epigenetic alteration(s) and the cancer(s) in which these epimutations are found. Review articles, [110] and broad experimental survey articles [111] [112] also document most of these epigenetic DNA repair deficiencies in cancers.

    Red-highlighted genes are frequently reduced or silenced by epigenetic mechanisms in various cancers. When these genes have low or absent expression, DNA damages can accumulate. Replication errors past these damages (see translesion synthesis) can lead to increased mutations and, ultimately, cancer. Epigenetic repression of DNA repair genes in accurate DNA repair pathways appear to be central to carcinogenesis.

    The two gray-highlighted genes RAD51 and BRCA2, are required for homologous recombinational repair. They are sometimes epigenetically over-expressed and sometimes under-expressed in certain cancers. As indicated in the Wikipedia articles on RAD51 and BRCA2, such cancers ordinarily have epigenetic deficiencies in other DNA repair genes. These repair deficiencies would likely cause increased unrepaired DNA damages. The over-expression of RAD51 and BRCA2 seen in these cancers may reflect selective pressures for compensatory RAD51 or BRCA2 over-expression and increased homologous recombinational repair to at least partially deal with such excess DNA damages. In those cases where RAD51 or BRCA2 are under-expressed, this would itself lead to increased unrepaired DNA damages. Replication errors past these damages (see translesion synthesis) could cause increased mutations and cancer, so that under-expression of RAD51 or BRCA2 would be carcinogenic in itself.

    Cyan-highlighted genes are in the microhomology-mediated end joining (MMEJ) pathway and are up-regulated in cancer. MMEJ is an additional error-prone inaccurate repair pathway for double-strand breaks. In MMEJ repair of a double-strand break, an homology of 5–25 complementary base pairs between both paired strands is sufficient to align the strands, but mismatched ends (flaps) are usually present. MMEJ removes the extra nucleotides (flaps) where strands are joined, and then ligates the strands to create an intact DNA double helix. MMEJ almost always involves at least a small deletion, so that it is a mutagenic pathway. [24] FEN1, the flap endonuclease in MMEJ, is epigenetically increased by promoter hypomethylation and is over-expressed in the majority of cancers of the breast, [113] prostate, [114] stomach, [115] [116] neuroblastomas, [117] pancreas, [118] and lung. [119] PARP1 is also over-expressed when its promoter region ETS site is epigenetically hypomethylated, and this contributes to progression to endometrial cancer [120] and BRCA-mutated serous ovarian cancer. [121] Other genes in the MMEJ pathway are also over-expressed in a number of cancers (see MMEJ for summary), and are also shown in cyan.

    Genome-wide distribution of DNA repair in human somatic cells Edit

    Differential activity of DNA repair pathways across various regions of the human genome causes mutations to be very unevenly distributed within tumor genomes. [122] [123] In particular, the gene-rich, early-replicating regions of the human genome exhibit lower mutation frequencies than the gene-poor, late-replicating heterochromatin. One mechanism underlying this involves the histone modification H3K36me3, which can recruit mismatch repair proteins, [124] thereby lowering mutation rates in H3K36me3-marked regions. [125] Another important mechanism concerns nucleotide excision repair, which can be recruited by the transcription machinery, lowering somatic mutation rates in active genes [123] and other open chromatin regions. [126]

    The basic processes of DNA repair are highly conserved among both prokaryotes and eukaryotes and even among bacteriophages (viruses which infect bacteria) however, more complex organisms with more complex genomes have correspondingly more complex repair mechanisms. [127] The ability of a large number of protein structural motifs to catalyze relevant chemical reactions has played a significant role in the elaboration of repair mechanisms during evolution. For an extremely detailed review of hypotheses relating to the evolution of DNA repair, see. [128]

    The fossil record indicates that single-cell life began to proliferate on the planet at some point during the Precambrian period, although exactly when recognizably modern life first emerged is unclear. Nucleic acids became the sole and universal means of encoding genetic information, requiring DNA repair mechanisms that in their basic form have been inherited by all extant life forms from their common ancestor. The emergence of Earth's oxygen-rich atmosphere (known as the "oxygen catastrophe") due to photosynthetic organisms, as well as the presence of potentially damaging free radicals in the cell due to oxidative phosphorylation, necessitated the evolution of DNA repair mechanisms that act specifically to counter the types of damage induced by oxidative stress.

    Rate of evolutionary change Edit

    On some occasions, DNA damage is not repaired, or is repaired by an error-prone mechanism that results in a change from the original sequence. When this occurs, mutations may propagate into the genomes of the cell's progeny. Should such an event occur in a germ line cell that will eventually produce a gamete, the mutation has the potential to be passed on to the organism's offspring. The rate of evolution in a particular species (or, in a particular gene) is a function of the rate of mutation. As a consequence, the rate and accuracy of DNA repair mechanisms have an influence over the process of evolutionary change. [129] DNA damage protection and repair does not influence the rate of adaptation by gene regulation and by recombination and selection of alleles. On the other hand, DNA damage repair and protection does influence the rate of accumulation of irreparable, advantageous, code expanding, inheritable mutations, and slows down the evolutionary mechanism for expansion of the genome of organisms with new functionalities. The tension between evolvability and mutation repair and protection needs further investigation.

    A technology named clustered regularly interspaced short palindromic repeat (shortened to CRISPR-Cas9) was discovered in 2012. The new technology allows anyone with molecular biology training to alter the genes of any species with precision, by inducing DNA damage at a specific point and then altering DNA repair mechanisms to insert new genes. [130] It is cheaper, more efficient, and more precise than other technologies. With the help of CRISPR–Cas9, parts of a genome can be edited by scientists by removing, adding, or altering parts in a DNA sequence.

    Role of Rad51 and DNA repair in cancer: A molecular perspective

    The maintenance of genome integrity is essential for any organism survival and for the inheritance of traits to offspring. To the purpose, cells have developed a complex DNA repair system to defend the genetic information against both endogenous and exogenous sources of damage. Accordingly, multiple repair pathways can be aroused from the diverse forms of DNA lesions, which can be effective per se or via crosstalk with others to complete the whole DNA repair process. Deficiencies in DNA healing resulting in faulty repair and/or prolonged DNA damage can lead to genes mutations, chromosome rearrangements, genomic instability, and finally carcinogenesis and/or cancer progression. Although it might seem paradoxical, at the same time such defects in DNA repair pathways may have therapeutic implications for potential clinical practice. Here we provide an overview of the main DNA repair pathways, with special focus on the role played by homologous repair and the RAD51 recombinase protein in the cellular DNA damage response. We next discuss the recombinase structure and function per se and in combination with all its principal mediators and regulators. Finally, we conclude with an analysis of the manifold roles that RAD51 plays in carcinogenesis, cancer progression and anticancer drug resistance, and conclude this work with a survey of the most promising therapeutic strategies aimed at targeting RAD51 in experimental oncology.

    DNA repair is essential for cell vitality, cell survival, and cancer prevention, yet cells’ ability to patch up damaged DNA declines with age for reasons not fully understood.

    Now, research led by scientists at Harvard Medical School (HMS) reveals a critical step in a molecular chain of events that allows cells to mend their broken DNA.

    The findings, to be published March 24 in Science, offer a critical insight into how and why the body’s ability to fix DNA dwindles over time and point to a previously unknown role for the signaling molecule NAD as a key regulator of protein-to-protein interactions in DNA repair. NAD, identified a century ago, is already known for its role as a controller of cell-damaging oxidation.

    Additionally, experiments conducted in mice show that treatment with the NAD precursor NMN mitigates age-related DNA damage and wards off DNA damage from radiation exposure.

    Unraveling the mysteries of aging

    The scientists caution that the effects of many therapeutic substances are often profoundly different in mice and humans owing to critical differences in biology. However, if affirmed in further animal studies and in humans, the findings can help pave the way to therapies that prevent DNA damage associated with aging and with cancer treatments that involve radiation exposure and some types of chemotherapy, which, along with killing tumors, can cause considerable DNA damage in healthy cells. Human trials with NMN are expected to begin within six months, the researchers said.

    “Our results unveil a key mechanism in cellular degeneration and aging, but beyond that they point to a therapeutic avenue to halt and reverse age-related and radiation-induced DNA damage,” said senior author David Sinclair, professor in the Department of Genetics at HMS, co-director of the Paul F. Glenn Center for the Biology of Aging, and professor at the University of New South Wales School of Medicine in Sydney.

    A previous study led by Sinclair showed that NMN reversed muscle aging in mice.

    A plot with many characters

    The investigators started by looking at a cast of proteins and molecules suspected to play a part in the cellular aging process. Some of them were well-known characters, others more enigmatic figures.

    The researchers already knew that NAD, which declines steadily with age, boosts the activity of the SIRT1 protein, which delays aging and extends life in yeast, flies, and mice. Both SIRT1 and PARP1, a protein known to control DNA repair, consume NAD in their work.

    Another protein, DBC1, one of the most abundant proteins in humans and found across life forms from bacteria to plants and animals, was a far murkier presence. Because DBC1 previously had been shown to inhibit vitality-boosting SIRT1, the researchers suspected DBC1 may also somehow interact with PARP1, given the similar roles PARP1 and SIRT1 play.

    “We thought if there is a connection between SIRT1 and DBC1, on one hand, and between SIRT1 and PARP1 on the other, then maybe PARP1 and DBC1 were also engaged in some sort of intracellular game,” said Jun Li, first author on the study and a research fellow in the Department of Genetics at HMS.

    To get a better sense of the chemical relationship among the three proteins, the scientists measured the molecular markers of protein-to-protein interaction inside human kidney cells. DBC1 and PARP1 bound powerfully to each other. However, when NAD levels increased, that bond was disrupted. The more NAD was present inside cells, the fewer molecular bonds PARP1 and DBC1 could form. When researchers inhibited NAD, the number of PARP1-DBC1 bonds went up. In other words, when NAD is plentiful, it prevents DBC1 from binding to PARP1 and meddling with its ability to mend damaged DNA.

    What this suggests, the researchers said, is that as NAD declines with age, fewer and fewer NAD molecules are around to stop the harmful interaction between DBC1 and PARP1. The result: DNA breaks go unrepaired and, as these breaks accumulate over time, precipitate cell damage, cell mutations, cell death, and loss of organ function.

    Averting mischief

    Next, to understand how exactly NAD prevents DBC1 from binding to PARP1, the team homed in on a region of DBC1 known as NHD, a pocket-like structure found in some 80,000 proteins across life forms and species whose function has eluded scientists. The team’s experiments showed that NHD is an NAD binding site and that in DBC1, NAD blocks this specific region to prevent DBC1 from locking in with PARP1 and interfering with DNA repair.

    Sinclair said that since NHD is so common across species, the finding suggests that by binding to it, NAD may play a similar role averting harmful protein interactions across many species to control DNA repair and other cell survival processes.


    Biology 171

    By the end of this section, you will be able to do the following:

    DNA replication is a highly accurate process, but mistakes can occasionally occur, such as a DNA polymerase inserting a wrong base. Uncorrected mistakes may sometimes lead to serious consequences, such as cancer. Repair mechanisms correct the mistakes. In rare cases, mistakes are not corrected, leading to mutations in other cases, repair enzymes are themselves mutated or defective.

    Most of the mistakes during DNA replication are promptly corrected by the proofreading ability of DNA polymerase itself. ((Figure)). In proofreading , the DNA pol reads the newly added base before adding the next one, so a correction can be made. The polymerase checks whether the newly added base has paired correctly with the base in the template strand. If it is the right base, the next nucleotide is added. If an incorrect base has been added, the enzyme makes a cut at the phosphodiester bond and releases the wrong nucleotide. This is performed by the 3′ exonuclease action of DNA pol. Once the incorrect nucleotide has been removed, it can be replaced by the correct one.

    Some errors are not corrected during replication, but are instead corrected after replication is completed this type of repair is known as mismatch repair ((Figure)). Specific repair enzymes recognize the mispaired nucleotide and excise part of the strand that contains it the excised region is then resynthesized. If the mismatch remains uncorrected, it may lead to more permanent damage when the mismatched DNA is replicated. How do mismatch repair enzymes recognize which of the two bases is the incorrect one? In E. coli, after replication, the nitrogenous base adenine acquires a methyl group the parental DNA strand will have methyl groups, whereas the newly synthesized strand lacks them. Thus, DNA polymerase is able to remove the wrongly incorporated bases from the newly synthesized, non-methylated strand. In eukaryotes, the mechanism is not very well understood, but it is believed to involve recognition of unsealed nicks in the new strand, as well as a short-term continuing association of some of the replication proteins with the new daughter strand after replication has completed.

    Another type of repair mechanism, nucleotide excision repair , is similar to mismatch repair, except that it is used to remove damaged bases rather than mismatched ones. The repair enzymes replace abnormal bases by making a cut on both the 3′ and 5′ ends of the damaged base ((Figure)). The segment of DNA is removed and replaced with the correctly paired nucleotides by the action of DNA pol. Once the bases are filled in, the remaining gap is sealed with a phosphodiester linkage catalyzed by DNA ligase. This repair mechanism is often employed when UV exposure causes the formation of pyrimidine dimers.

    A well-studied example of mistakes not being corrected is seen in people suffering from xeroderma pigmentosa ((Figure)). Affected individuals have skin that is highly sensitive to UV rays from the sun. When individuals are exposed to UV light, pyrimidine dimers, especially those of thymine, are formed people with xeroderma pigmentosa are not able to repair the damage. These are not repaired because of a defect in the nucleotide excision repair enzymes, whereas in normal individuals, the thymine dimers are excised and the defect is corrected. The thymine dimers distort the structure of the DNA double helix, and this may cause problems during DNA replication. People with xeroderma pigmentosa may have a higher risk of contracting skin cancer than those who don’t have the condition.

    Errors during DNA replication are not the only reason why mutations arise in DNA. Mutations , variations in the nucleotide sequence of a genome, can also occur because of damage to DNA. Such mutations may be of two types: induced or spontaneous. Induced mutations are those that result from an exposure to chemicals, UV rays, x-rays, or some other environmental agent. Spontaneous mutations occur without any exposure to any environmental agent they are a result of natural reactions taking place within the body.

    Mutations may have a wide range of effects. Point mutations are those mutations that affect a single base pair. The most common nucleotide mutations are substitutions, in which one base is replaced by another. These substitutions can be of two types, either transitions or transversions. Transition substitution refers to a purine or pyrimidine being replaced by a base of the same kind for example, a purine such as adenine may be replaced by the purine guanine. Transversion substitution refers to a purine being replaced by a pyrimidine, or vice versa for example, cytosine, a pyrimidine, is replaced by adenine, a purine. Some point mutations are not expressed these are known as silent mutations. Silent mutations are usually due to a substitution in the third base of a codon, which often represents the same amino acid as the original codon. Other point mutations can result in the replacement of one amino acid by another, which may alter the function of the protein. Point mutations that generate a stop codon can terminate a protein early.

    Some mutations can result in an increased number of copies of the same codon. These are called trinucleotide repeat expansions and result in repeated regions of the same amino acid. Mutations can also be the result of the addition of a base, known as an insertion, or the removal of a base, also known as deletion. If an insertion or deletion results in the alteration of the translational reading frame (a frameshift mutation), the resultant protein is usually nonfunctional. Sometimes a piece of DNA from one chromosome may get translocated to another chromosome or to another region of the same chromosome this is also known as translocation. These mutation types are shown in (Figure).

    A frameshift mutation that results in the insertion of three nucleotides is often less deleterious than a mutation that results in the insertion of one nucleotide. Why?

    Mutations in repair genes have been known to cause cancer. Many mutated repair genes have been implicated in certain forms of pancreatic cancer, colon cancer, and colorectal cancer. Mutations can affect either somatic cells or germ cells. If many mutations accumulate in a somatic cell, they may lead to problems such as the uncontrolled cell division observed in cancer. If a mutation takes place in germ cells, the mutation will be passed on to the next generation, as in the case of hemophilia and xeroderma pigmentosa.

    Section Summary

    DNA polymerase can make mistakes while adding nucleotides. It edits the DNA by proofreading every newly added base. Incorrect bases are removed and replaced by the correct base before proceeding with elongation. Most mistakes are corrected during replication, although when this does not happen, the mismatch repair mechanism is employed. Mismatch repair enzymes recognize the wrongly incorporated base and excise it from the DNA, replacing it with the correct base. In yet another type of repair, nucleotide excision repair, a damaged base is removed along with a few bases on the 5′ and 3′ end, and these are replaced by copying the template with the help of DNA polymerase. The ends of the newly synthesized fragment are attached to the rest of the DNA using DNA ligase, which creates a phosphodiester bond.

    Most mistakes are corrected, and if they are not, they may result in a mutation, defined as a permanent change in the DNA sequence. Mutations can be of many types, such as substitution, deletion, insertion, and trinucleotide repeat expansions. Mutations in repair genes may lead to serious consequences such as cancer. Mutations can be induced or may occur spontaneously.

    Art Connections

    (Figure) A frameshift mutation that results in the insertion of three nucleotides is often less deleterious than a mutation that results in the insertion of one nucleotide. Why?

    (Figure) If three nucleotides are added, one additional amino acid will be incorporated into the protein chain, but the reading frame wont shift.

    Free Response

    What is the consequence of mutation of a mismatch repair enzyme? How will this affect the function of a gene?

    Mutations are not repaired, as in the case of xeroderma pigmentosa. Gene function may be affected or it may not be expressed.

    An adult with a history of tanning has his genome sequenced. The beginning of a protein-coding region of his DNA reads ATGGGGATATGGCAT. If the protein-coding region of a healthy adult reads ATGGGGATATGAGCAT, identify the site and type of mutation.

    This is a frameshift mutation with a deletion of an “A” in the 12 th position of the coding region.


    Watch the video: What happens when your DNA is damaged? - Monica Menesini (May 2022).