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5.5: DNA Replication - Biology

5.5: DNA Replication - Biology


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With their multiple origins, how does the eukaryotic cell know which origins have been already replicated and which still await replication?

An observation: When a cell in G2 of the cell cycle is fused with a cell in S phase, the DNA of the G2 nucleus does not begin replicating again even though replication is proceeding normally in the S-phase nucleus. Not until mitosis is completed, can freshly-synthesized DNA be replicated again.

Two control mechanisms have been identified — one positive and one negative. This redundancy probably reflects the crucial importance of precise replication to the integrity of the genome.

Licensing: positive control of replication

In order to be replicated, each origin of replication must be bound by:

  • an Origin Recognition Complex of proteins (ORC). These remain on the DNA throughout the process.
  • Accessory proteins called licensing factors. These accumulate in the nucleus during G1 of the cell cycle. They include:
    • Cdc-6 and Cdt-1, which bind to the ORC and are essential for coating the DNA with
    • MCM proteins. Only DNA coated with MCM proteins (there are 6 of them) can be replicated.

Once replication begins in S phase,

  • Cdc-6 and Cdt-1 leave the ORCs (the latter by ubiquination and destruction in proteasomes).
  • The MCM proteins leave in front of the advancing replication fork.

Effects of DNA replication on mRNA noise

Gene expression noise affects a cell’s biological state and contributes to such phenomena as phenotype switching and cell fate determination. By examining chromosome replication—which is tightly controlled and thus exhibits little noise—we show that variability in mRNA levels across a population is less than previously expected. This noise is due to the transient relaxation of the mRNA from a low- to a high-copy steady state after a gene replication event. Gene location, mRNA degradation rate, and cell doubling time all contribute to observed noise. Our results demonstrate that it is essential to account for gene replication when modelling gene expression or when interpreting experimental results.


What is PCR

PCR (polymerase chain reaction) is a widely used molecular biological technique to produce thousands to millions of copies of a particular DNA fragment through the exponential amplification. It was developed by Kary Mullis in 1983. The most significant feature of PCR is that it relies on thermal cycling. Therefore, it permits different temperature-dependent reactions to occur, including DNA melting and enzyme-driven DNA polymerization. On the other hand, the two main reagents used in PCR are the DNA primers, which are complementary to the target sequence and a heat-stable DNA polymerase such as Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus. Meanwhile, the forward and reverse PCR primers flank the region to be polymerized on the DNA fragment.

Figure 1: Polymerase Chain Reaction

Furthermore, the three main steps involved in a PCR are:

  1. Denaturation – Melting DNA duplex into two single strands by heating to 94-95 °C.
  2. Annealing – The binding of the forward and reverse primers to the complementary sequences on the template. The temperature of this step depends on the melting temperature of the primer combination.
  3. Primer extension – DNA polymerase enzyme extends each of the primers at their 3’end by adding complementary bases to the growing strand. The optimum temperature of Taq polymerase, 72 °C is used as the temperature in the extension step. The time of the extension depends on the number of base pairs in the template strand.

Generally, the three steps are repeated for 30-40 times during the PCR to obtain an exponential growth of the DNA fragment of interest.


A scientist puts nucleotide chains of UUUUUU in a test tube under conditions allowing protein synthesis. Soon the test tube is full of polypeptide chains composed only one the amino acid phenylalanine. What does this experiment indicate?

A. The amino acid phenylalanine is composed of uracil.

B. UUU codes for the amino acid phenylalanine.

C. Protein synthesis malfunctions in test tubes.

D. Most proteins contain only one type of amino acid.


Abstract

The CDC7 kinase, by phosphorylating the MCM DNA helicase, is a key switch for DNA replication initiation. ATP competitive CDC7 inhibitors are being developed as potential anticancer agents however how human cells respond to the selective pharmacological inhibition of this kinase is controversial and not understood. Here we have characterized the mode of action of the two widely used CDC7 inhibitors, PHA-767491 and XL-413, which have become important tool compounds to explore the kinase’s cellular functions. We have used a chemical genetics approach to further characterize pharmacological CDC7 inhibition and CRISPR/CAS9 technology to assess the requirement for kinase activity for cell proliferation. We show that, in human breast cells, CDC7 is essential and that CDC7 kinase activity is formally required for proliferation. However, full and sustained inhibition of the kinase, which is required to block the cell-cycle progression with ATP competitor compounds, is problematic to achieve. We establish that MCM2 phosphorylation is highly sensitive to CDC7 inhibition and, as a biomarker, it lacks in dynamic range since it is easily lost at concentrations of inhibitors that only mildly affect DNA synthesis. Furthermore, we find that the cellular effects of selective CDC7 inhibitors can be altered by the concomitant inhibition of cell-cycle and transcriptional CDKs. This work shows that DNA replication and cell proliferation can occur with reduced CDC7 activity for at least 5 days and that the bulk of DNA synthesis is not tightly coupled to MCM2 phosphorylation and provides guidance for the development of next generation CDC7 inhibitors.


What is transcription?

It involves making a copy of DNA into RNA. The part of DNA that codes for genes is copied into the messenger RNA. The two strands of DNA helix are unwind and separated. The RNA polymerase, a special type of enzyme travels along the strands of DNA and binds RNA nucleotides to it until it forms a complete strand of messenger RNA.

The messenger RNA also known as mRNA is the cell’s blueprint, which is used to construct a specific type of protein. The mRNA travels from the nucleus to the cytoplasm where the gene expression takes place. (1, 2, 3, and 4)

Image 2: An image presentation on how DNA replication takes place.

Picture Source:wikimedia.org


Replication redux

We propose an alternative definition for replication that is more inclusive of all research and more relevant for the role of replication in advancing knowledge. Replication is a study for which any outcome would be considered diagnostic evidence about a claim from prior research. This definition reduces emphasis on operational characteristics of the study and increases emphasis on the interpretation of possible outcomes.

To be a replication, 2 things must be true: outcomes consistent with a prior claim would increase confidence in the claim, and outcomes inconsistent with a prior claim would decrease confidence in the claim. The symmetry promotes replication as a mechanism for confronting prior claims with new evidence. Therefore, declaring that a study is a replication is a theoretical commitment. Replication provides the opportunity to test whether existing theories, hypotheses, or models are able to predict outcomes that have not yet been observed. Successful replications increase confidence in those models unsuccessful replications decrease confidence and spur theoretical innovation to improve or discard the model. This does not imply that the magnitude of belief change is symmetrical for “successes” and “failures.” Prior and existing evidence inform the extent to which replication outcomes alter beliefs. However, as a theoretical commitment, replication does imply precommitment to taking all outcomes seriously.

Because replication is defined based on theoretical expectations, not everyone will agree that one study is a replication of another. Moreover, it is not always possible to make precommitments to the diagnosticity of a study as a replication, often for the simple reason that study outcomes are already known. Deciding whether studies are replications after observing the outcomes can leverage post hoc reasoning biases to dismiss “failures” as nonreplications and “successes” as diagnostic tests of the claims, or the reverse if the observer wishes to discredit the claims. This can unproductively retard research progress by dismissing replication counterevidence. Simultaneously, replications can fail to meet their intended diagnostic aims because of error or malfunction in the procedure that is only identifiable after the fact. When there is uncertainty about the status of claims and the quality of methods, there is no easy solution to distinguishing between motivated and principled reasoning about evidence. Science’s most effective solution is to replicate, again.

At its best, science minimizes the impact of ideological commitments and reasoning biases by being an open, social enterprise. To achieve that, researchers should be rewarded for articulating their theories clearly and a priori so that they can be productively confronted with evidence [4,6]. Better theories are those that make it clear how they can be supported and challenged by replication. Repeated replication is often necessary to resolve confidence in a claim, and, invariably, researchers will have plenty to argue about even when replication and precommitment are normative practices.


Mechanism of Replication (Basic)

Knowing the structure of DNA, scientists speculated and then proved that DNA is the template for copying the genetic code. See how information in DNA is copied to make new DNA molecules.

Duration: 1 minutes, 5 seconds

Using computer animation based on molecular research, we are now able to see how DNA is actually Using computer animation based on molecular research, we are now able to see how DNA is actually copied in living cells. You are looking at an assembly line of amazing miniature biochemical machines that are pulling apart the DNA double helix and cranking out a copy of each strand. The DNA to be copied enters the production line from bottom left. The whirling blue molecular machine is called helicase. It spins the DNA as fast as a jet engine as it unwinds the double helix into two strands. One strand is copied continuously and can be seen spooling off to the right. Things are not so simple for the other strand because it must be copied backwards. It is drawn out repeatedly in loops, and copied one section at a time. The end result is two new DNA molecules.

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5.5: DNA Replication - Biology

During cell division, each new cell or daughter cell must have the same number of chromosomes as the parent cell. Therefore when a cell divides, the chromosomes split lengthways and each half enters the new cell ensuring the same number is in the new cell and parent cell.

Chromosomes are made up of DNA, so in order for the chromosomes to make exact copies of themselves so must the DNA. If this was not possible, the new cells would not hold all the correct genetic information they require (remember a gene is a small section of DNA).

DNA replication is a complicated process involving enzymes and proteins contained within the cell. The details of this process are beyond the scope of GCSEs so a summary of the main steps is provided to give a simplified overview of the process.

  1. The DNA molecule unwinds by the action of an enzyme which beaks the hydrogen bonds holding the base pairs together.
  2. Small proteins bind to each side to keep the two strands separated.
  3. New nucleotides, which are present in the nucleus of the cell, line up along each single DNA strand following the base pair rules.
    A lines up alongside T
    C lines up alongside G
  4. The nucleotides join together making two new DNA molecules, and each one is an identical copy of the parent cell’s DNA. The DNA molecules automatically wind up into the double helix shape.

As the DNA is such a long molecule the replication process begins at multiple locations along the molecule.


Recap and Conclusion

Hopefully this information did not make your head spin. If it did, below you will find a short recap. Both molecules contain a phosphate backbone and are made up of nucleotides. DNA carries all the information needed for DNA replication and transfer new information to new cells. This information is also needed to make proteins the body needs for various purposes including regulation of DNA replication. RNA is transcribed from the DNA to make these proteins (the central dogma, Figure 1). RNA is transcribed and processed within the nucleus, it then moves through the nuclear pores for protein translation in the cytoplasm. In this sense, DNA and RNA are the perfect partners in crime. What DNA can’t do, RNA can and what DNA can do RNA can’t. What results from this perfect partnership is that the single-stranded RNA can be made from the double stranded DNA. The nucleus confined DNA can send its message to the rest of the cell with the aid of the RNA, which moves around freely through the cell. The “dangers” faced by the RNA means it might or does need to be recreated and continuously destroyed, DNA provides the platform for the rebirth of this molecule. By all accounts, DNA and RNA differ in just the right amount while they are also similar just right and hopefully this point was made plenty clear here.

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1 thought on &ldquoWhat are the similarities between DNA and RNA&rdquo

This is a very clear and effective discussion especially for a relative novice ( yours truly).
I have a clearer understanding of the relative significance which I find to be enlightening and exciting.
In my case ( approaching 80 years old) the stepping stone process of your clarification makes for an eye opening revelation. Love this stuff! Want my grandchildren to experience your elucidating explanation.

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Watch the video: From DNA to protein - 3D (May 2022).