Crossing over : Holliday model

Crossing over : Holliday model

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In this figure out of the two heteroduplexes formed one is considered to be non-recombinant product(left) and the other recombinant product(right). My question is despite the exchange of middle DNA segment why is the right heteroduplex considered a non-recombinant product or patch. I've been reading two books:

  1. Molecular Biology: Principles of Genome Function By Nancy Lynn Craig, Rachel Green, Orna Cohen-Fix, Carol Greider, Gisela Storz, Cynthia
  2. Molecular Biology By Burton E. Tropp

both had the same illustration and descrpition.

Answer in brief

Heteroduplex called patch does not have double-stranded recombinant regions, there is a new fragment in a single strand only. It means that the two strands are not complementary at that regions and as soon as DNA repair systems detects it, the mistake will be corrected. In case of heteroduplex called splice, there are double-stranded changes, which do not go against the complementarity principle and thus, they will not be removed or corrected in any way.

Diagrammatic Explanation

After horizontal and vertical resolution patch and splice are formed respectively.

A better version of this diagram with complimentary base pairing is shown below $-$

In patch the heteroduplexes formed have only a single stranded middle recombinant region which is not complimentary to the other strand when this is detected by the DNA repair system and corrected the duplexes formed are same as the original duplexes (before crossing over) so the products of horizontal resolution is called non-recombinant products.

In splice the heteroduplexes formed have double stranded recombinant segments with middle non complementary region which when detected and fixed by the cell's DNA repair system two duplexes are formed having recombinant DNA segments, so they are together called recombinant products. The recombination in splice is well represented by the reversal of terminal genes.

Robin Holliday 1932–2014

It takes an extraordinary talent to accurately describe a biological process from limited available data. It is more remarkable when charisma is combined with the ability to identify key fundamental questions that pave the path for entirely new research areas. Robin Holliday possessed these distinguishing traits and survived to see several of his proposed models withstand decades of experimental scrutiny and remain as the founding principles for various scientific fields. Robin died peacefully on 9 April 2014 in Sydney.

Robin, a British national, was born in the British Mandate of Palestine in 1932. He spent his childhood and adolescence in Sri Lanka, South Africa and Gibraltar before returning to Britain to complete his secondary education. He continued his studies at Cambridge University, where he graduated in 1955 with First Class Honours in Natural Sciences. After a successful career in the UK, Robin moved in 1988 to Sydney, where he continued his research until his retirement in 1997.

Robin started his research career at the John Innes Institute in 1958, only three years after the structure of DNA was revealed by Watson and Crick to be a double-stranded helix. During his PhD work and early research career, he established genetic experimental models using the parasitic dimorphic fungus Ustilago maydis. These included the isolation of the first eukaryotic mutants in DNA repair and recombination, which established the proof of principle that such mutants could be obtained from genetic screens. Robin's own research, as well as earlier studies on fine-structure genetic mapping, strongly suggested that DNA molecules must be capable of recombining with homologous partners.

Robin proposed a molecular model, based on the insight provided by these studies, to describe the process of homologous recombination. In this model, unraveling DNA strands anneal with complementary bases in opposite partners, thus accounting for the genetic phenomenon of crossing over. Robin went on to suggest that if the crossover occurred at a DNA site where the parental molecules differed, the mismatched region would be corrected, and this would lead to gene conversion, a form of inheritance that does not follow Mendel's laws. He proposed that the point at which the strands exchanged partners would be the site of DNA recombination. Robin predicted that the DNA structure at this exchange point would be a symmetrical four-way junction that could be resolved in either of two orientations, to yield DNA molecules with distal genetic markers in the recombinant or the parental configuration. Publication of this model stimulated discussion and experimentation worldwide, and the model became known as the Holliday model. Its major predictions, including the formation of the symmetrical four-way intermediate, now known as the Holliday junction, were confirmed experimentally in numerous laboratories. The Holliday model has been elaborated upon and amended over the past five decades to accommodate and explain new genetic data, but the four-way Holliday junction is universally recognized as the central intermediate structure underpinning genetic recombination in all domains of life.

In 1965, Robin moved to the National Institute for Medical Research in Mill Hill, London. Three years later, he was promoted to head the Genetics Division, which grew dramatically under his leadership and thrived as a highly successful research environment. Here, Robin expanded his research interests to encompass epigenetics and aging. In 1975, Robin and his student John Pugh proposed a model to explain how the pattern of gene expression between tissues or cell types differs, even though cells share the same DNA. They predicted that epigenetic mechanisms silence groups of genes and that the pattern of such silencing should be heritable. Their molecular model for the heritable switching of gene activities was based on the methylation of C residues in DNA. This model, based entirely on theoretical considerations, suggested that DNA methylation would control gene expression and establish tissue-specific expression profiles during development. Further, to account for the heritability of methylation, they predicted the existence of a maintenance DNA methyltransferase that would recognize hemimethylated DNA shortly after replication and copy the pattern on the newly synthesized strand. The proposal that methylation causes gene silencing proved to be correct, and the model is now supported by substantial evidence, some of which emerged from Robin's own studies on epigenetic regulation. Moreover, maintenance DNA methyltransferase enzymes have been identified and characterized, proving that heritable patterns of DNA methylation indeed provide a mechanism to regulate developmental processes.

As understanding of the importance of maintaining genome integrity grew, the close interface between cellular and organismal aging came to be appreciated. This represented a third area of research in which Robin made notable contributions. Through longstanding association with Leslie Orgel, Robin played a leading part in testing Orgel's hypothesis that cyclical feedback of errors in protein synthesis might have a fundamental role in aging. He used an array of biological models, from Escherichia coli to primary human cells in culture, to address the mechanisms of cellular senescence and aging. Robin's interest in the molecular causes of aging helped to spotlight this crucial problem in biology.

At the National Institute for Medical Research, Robin led a team of researchers that made important contributions to all the areas mentioned above. He trained many scientists who have progressed to run their own laboratories or pursued other careers within science. During this time, he also hosted many visiting scientists from around the world. For many of us, the learning experience at the Genetics Division occurred at multiple levels, including philosophical discussions during coffee breaks on such diverse questions as the evolution of morals. Robin often led these discussions, which provided a profound perspective on everyday research and emerging scientific questions. For the younger students, there was constant intellectual stimulation, with Robin insisting that we should never stop thinking about the relevance of our findings to the fundamental principles of life. In this way, Robin endowed us with a passion for science.

Robin was a prolific writer, continuing to publish well after his retirement. In addition to research publications, he wrote a large number of reviews covering scientific as well as historical reflections. He was a Fellow of the Royal Society, a member of the European Molecular Biology Organization, a member of the Australian Academy of Science and a Foreign Fellow of the Indian National Science Academy. He received prestigious awards including the Royal Medal, one of the Royal Society's premier awards, highlighting excellence in science, and the Lord Cohen Medal for Gerontological Research. Finally, Robin was also an outstanding sculptor, with many of his sculptures inspired by his biological studies. Some of his sculptures are now on display in scientific institutions including the Royal Society, the Laboratory of Molecular Biology in Cambridge, UK and the Genome Damage and Stability Centre in the University of Sussex.

Robin mentored us to the end of his life. His last advice to Fotini, two weeks before his death was: “Do good science, and don't worry about the rest.” How characteristic of Robin, and how priceless this advice will always be.


Meiosis is the specialised reductive division that generates haploid cells. During this process, a single round of replication is followed by two rounds of chromosome segregation: in the first division (meiosis I), homologous chromosomes segregate, whereas in the second division, sister chromatids segregate (meiosis II). A key step in meiosis I is the recognition of homologous chromosomes, which then align and pair along the length of the chromosome. Once homologues have aligned, synapsis can proceed with the formation of the synaptonemal complex (SC), a protein structure that supports and maintains homologues in close juxtaposition and serves as a scaffold for crossover-promoting recombination factors. Meiotic crossing over involves the generation of meiotic double-strand breaks (DSBs), which are subsequently repaired either as crossovers or non-crossovers (Fig. 1). Meiotic recombination is not only necessary to create new allele combinations that generate genetic diversity, but is also essential in ensuring accurate chromosome segregation at the first meiotic division because the crossover acts as a tether between homologues, which ensures that each homologue will properly align at the metaphase plate and thereby correctly attach to the spindle. DSB repair occurs concurrently with SC formation and is required for normal synapsis in yeast and mice (Baudat et al., 2000 Roeder, 1997 Romanienko and Camerini-Otero, 2000), whereas in Caenorhabditis elegans and Drosophila melanogaster, homologue pairing and SC formation can occur independently of meiotic recombination (Colaiacovo et al., 2003 Dernburg et al., 1998 Liu et al., 2002 McKim et al., 1998).

The process of meiotic recombination is initiated when meiotic DSBs are created by the endonuclease SPO11, in conjunction with a number of additional proteins (Keeney and Neale, 2006). DSBs are then resected to generate 3′ single-strand DNA (ssDNA) overhangs that are initially bound by replication protein A (RPA), which is subsequently displaced by the recombinase radiation sensitive 51 (RAD51) and/or the meiosis-specific recombinase dosage suppressor of Mck1 (DMC1) to form nucleoprotein filaments. These filaments serve to find a complimentary sequence within a homologous chromosome, at which they instigate single-end strand invasions (Hunter and Kleckner, 2001) to generate so-called displacement loop (D loop) recombination intermediates (Fig. 1). If the second end of the original DSB binds with the homologous chromosome, a double Holliday junction is formed, which can be resolved to generate either a non-crossover or an interhomologue crossover, the latter of which is hereafter referred to simply as crossover (Bishop and Zickler, 2004 Schwacha and Kleckner, 1995). Double Holliday junctions can also be processed through dissolution, which results in a non-crossover (Fig. 1) (Wu and Hickson, 2003). Meiotic non-crossovers have also been proposed to form when strand invasion is transient, and when a limited amount of DNA synthesis occurs before the invaded strand dissociates and anneals to its partner strand, as in mitotic synthesis-dependent strand annealing (SDSA see Box 1 and Fig. 1) (Allers and Lichten, 2001 Bishop and Zickler, 2004). During meiosis in budding yeast, non-crossover heteroduplex products are found to form with the same timing as double Holliday junctions, whereas crossovers occur later (Allers and Lichten, 2001), consistent with the idea that crossovers and non-crossovers are formed through distinct pathways. Analysis of ZMM mutants (Zip1, Zip2, Zip3, Zip4, Mer3, Msh4 and Msh5 further discussed below) in yeast indicates that the decision between crossover and non-crossover is made very early, i.e. at or prior to the establishment of a stable single-end invasion intermediate, when one of the two ends of the DSB invades its homologous chromatid (Bishop and Zickler, 2004 Borner et al., 2004 Hunter and Kleckner, 2001). Thus, the crossover–non-crossover decision is thought to occur around the time of strand exchange.

Model for meiotic crossover or non-crossover formation. Double strand breaks are generated and their 5′ ends are resected to generate a 3′ overhang. A strand invasion event then generates a single-end invasion D loop intermediate. If the second end of the original DSB also engages with the homologue, a double Holliday junction is formed (shown on the left). The double Holliday junction can be resolved to form either a crossover (interference-dependent) or a non-crossover. Alternatively, the junction can be dissolved by double Holliday junction dissolution to form a non-crossover. Instead of forming a double Holliday junction, the D loop can be dissociated and the invading strand can associate with the opposite end of the original break, as in synthesis-dependent strand annealing (SDSA), to form a non-crossover. Alternatively, the intermediate can be acted upon by enzymes such as Mus81 that can form interference-independent crossovers.

Model for meiotic crossover or non-crossover formation. Double strand breaks are generated and their 5′ ends are resected to generate a 3′ overhang. A strand invasion event then generates a single-end invasion D loop intermediate. If the second end of the original DSB also engages with the homologue, a double Holliday junction is formed (shown on the left). The double Holliday junction can be resolved to form either a crossover (interference-dependent) or a non-crossover. Alternatively, the junction can be dissolved by double Holliday junction dissolution to form a non-crossover. Instead of forming a double Holliday junction, the D loop can be dissociated and the invading strand can associate with the opposite end of the original break, as in synthesis-dependent strand annealing (SDSA), to form a non-crossover. Alternatively, the intermediate can be acted upon by enzymes such as Mus81 that can form interference-independent crossovers.

This Commentary will discuss the factors that contribute to crossover or non-crossover formation in meiosis, including the generation and positioning of meiotic DSBs, formation of the SC and generation of recombination intermediates. We will also discuss how interhomologue crossing over is promoted compared with intersister repair, how recombination intermediates are resolved into crossovers as well as how anti-recombinases prevent crossing over. Crossover interference, assurance and homeostasis will also be discussed (see text box), including a summary of the current models for how crossover interference is established.

Holliday junction

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Holliday junction, cross-shaped structure that forms during the process of genetic recombination, when two double-stranded DNA molecules become separated into four strands in order to exchange segments of genetic information. This structure is named after British geneticist Robin Holliday, who proposed the original model for homologous (or general) recombination in 1964.

Homologous recombination occurs during meiosis and is characterized by the exchange of genes between a maternal chromatid and a paternal chromatid of a homologous chromosome pair. The two parent DNA molecules, which have long stretches of similar base sequences, are separated into single strands, resulting in base pairing that leads to a four-stranded DNA structure. The Holliday junction travels along the DNA duplex by “unzipping” one strand and reforming the hydrogen bonds on the second strand.

This article was most recently revised and updated by Kara Rogers, Senior Editor.

Anticrossover and Procrossover Activities of Sgs1

Studies of anticrossover helicases have been particularly illuminating. Crossovers are a beneficial product of meiotic recombination, but they are avoided during mitotic recombination because they can cause genome instability. Double-strand breaks in nonmeiotic cells are preferentially repaired into noncrossovers, largely through the action of various anticrossover helicases. One key anticrossover protein is the Bloom syndrome helicase, BLM (reviewed in Andersen and Sekelsky 2010). Although BLM likely has many anticrossover functions, two activities are relevant to double-strand break repair. First, studies in Drosophila suggested that BLM promotes synthesis-dependent strand annealing, probably by disrupting D-loops after repair DNA synthesis (Adams et al. 2003 McVey et al. 2004), an activity human BLM has been shown to have in vitro (van Brabant et al. 2000 Bachrati et al. 2006). Second, in vitro studies demonstrated that BLM, together with topoisomerase IIIα and other proteins, can catalyze double-Holliday junction dissolution, a process in which the two Holliday junctions are migrated toward one another and then decatenated (Wu and Hickson 2003). Unlike resolution of double-Holliday junctions by cleavage, dissolution generates only noncrossovers.

Genetic studies suggested a similar anticrossover role for the S. cerevisiae BLM ortholog Sgs1 in meiosis. Crossovers are reduced in mutants lacking ZMM proteins, including Msh4–Msh5, but, remarkably, crossovers are restored in double mutants that also lack Sgs1 (Jessop et al. 2006 Oh et al. 2007). An attractive interpretation of these results is that one function of ZMMs is to antagonize the anticrossover activity of Sgs1. Thus, Msh4–Msh5 is an anti-anticrossover protein.

Although these experiments with ZMMs and Sgs1 are consistent with the known mitotic anticrossover functions of Sgs1, sgs1 mutants have only a modest increase in meiotic crossovers, much less than would be expected if all double-strand breaks were processed through a pathway in which double-Holliday junctions were produced and resolved into crossovers (Rockmill et al. 2003 Jessop et al. 2006). Novel insights into the solution to this apparent paradox came again from physical measurements of recombination intermediates and products. De Muyt et al. (2012) found that noncrossovers are still produced in sgs1 mutants, but, unlike the case in wild-type cells, these noncrossovers arise as joint molecules disappear and crossovers appear. This suggests that when Sgs1 is absent, double-Holliday junctions are resolved into crossovers and noncrossovers, as in the original double-strand break repair model.

Additional insights came from physical studies of recombination in mutants lacking the known Holliday junction resolvases. Three proteins, Mus81–Mms4, Yen1, and Slx1–Slx4, possess resolvase activity in vitro (Boddy et al. 2001 Ip et al. 2008 Fekairi et al. 2009). Mus81–Mms4 was shown to be important in generating mitotic crossovers, with Yen1 playing a compensatory or partially redundant role (Ho et al. 2010). Experiments by De Muyt et al. (2012) and Zakharyevich et al. (2012) found that single mutants lacking any one of these enzymes were still able to resolve most joint molecules and produce approximately normal numbers of crossovers. Even triple mutants lacking all three resolvases showed only a modest reduction in joint molecule resolution and crossover formation. These results suggest that the known resolvases collectively process only a small fraction of joint molecules. If these are joint molecules from the class II pathway, then most joint molecules must be generated in the class I pathway and be resolved by an unidentified resolvase.

Yet another surprise came when the same experiments were done in the absence of Sgs1. In this case, removing all three resolvases resulted in most joint molecules being left unresolved. Again, this result indicates that joint molecules produced in the absence of Sgs1 are different from those produced in the presence of Sgs1. In the absence of Sgs1, joint molecules are acted on by the known resolvases to produce both crossovers and noncrossovers, much like in the original double-strand break repair model. The known resolvases, functioning in the class II pathway, are therefore neither procrossover nor anticrossover, since they generate both outcomes. Conversely, joint molecules produced in the presence of Sgs1 (class I pathway) are cut by an unknown, procrossover resolvase to produce exclusively crossovers.

What is the identity of the procrossover resolvase that functions in the class I pathway? It had previously been suggested that the mismatch repair proteins Mlh1–Mlh3 (MutLγ complex) and Exo1 might act in double-Holliday junction resolution (Nishant et al. 2008 Zakharyevich et al. 2010). Crossovers are reduced in mlh3 mutants, but removal of Sgs1 restores crossovers, suggesting that Mlh3, like ZMMs, functions in the class I pathway (Oh et al. 2007). Consistent with this hypothesis, Zakharyevich et al. (2012) found that when all three known resolvases were removed, eliminating Mlh3 resulted in a similar reduction in crossovers as eliminating Sgs1. A parallel set of experiments suggested that Exo1 functions in a different pathway than Mus81–Mms4, putting Exo1 also in the class I pathway.

These results are consistent with Sgs1 having the expected anticrossover functions: It promotes synthesis-dependent strand annealing (in wild-type cells) and double-Holliday junction dissolution (when the three known resolvases and the putative procrossover resolvase are all missing). Unexpectedly, the results also reveal a procrossover role of Sgs1. This procrossover role may be in influencing pathway choice: In the presence of Sgs1, the ZMM-dependent class I crossover pathway can be used, but in the absence of Sgs1, the alternative class II pathway gives rise to both crossovers and noncrossovers from double-Holliday junction resolution.

Holliday model

A conceptualization of general genetic recombination of newly replicated DNA during meiosis. An endonuclease nicks one strand of each of the two daughter duplexes at homologous sites which permits them to cross-over before being ligated. branch migration of the crossing-over point, the Holliday junction, along the strands is also possible before they separate to form the recombinant products chi-forms (-shaped structures) are generated as the strands separate. In replication of closed circular (cc)DNA, figure-of-eight forms are generated as the two daughter ccDNAs separate, except where they have crossed-over and still adhere to each other. (see also double-strand-break repair model Meselson-Radding model)Strauss, B.S. (1992) Chemtracts Biochem. Mol. Biol. 3, 40-44 Shinagawa, H. and Iwasaki, H. (1996) Trends Biochem. Sci. 21, 107-111

If you know of any terms that have been omitted from this glossary that you feel would be useful to include, please send details to the Editorial Office at GenScript:

Crossing over : Holliday model - Biology

Während es im letzten Post um den Reparatur-Aspekt unserer Mutanten ging, soll im nächsten deren Beteiligung an der DNA-Rekombination betrachtet werden. Doch das wird ohne ein wenig Hintergrundwissen zur homologen Rekombination nicht so leicht werden, deshalb habe ich mich für diesen hoffentlich nicht zu komplexen Einschub entschieden.

Rekombination bedeutet zunächst, dass genetische Information zwischen zwei DNA-Molekülen ausgetauscht wird. Von den vielen Rekombinationsmechanismen ist die homologe Rekombination der konservativste. Das war jetzt keine politische Aussage, es bedeutet nur, dass es dabei idealerweise zu keiner Änderung der Sequenz kommt. Dies ist möglich, weil für die homologe Rekombination identische (= homologe) DNA-Sequenzen verwendet werden. Diese Sequenzen kommen bei diploiden Organismen wie dem Menschen oder Arabidopsis vom homologen Chromosom. Oder, noch besser, nach der Verdopplung der Chromosomen während der Replikation, vom Schwesterchromatid. Ausgangspunkt der homologen Rekombination ist immer ein Doppelstrangbruch. Dieser kann gewollt sein, etwa in der Meiose (dazu in einem anderen Teil der Paperübersicht mehr, wahrscheinlich #5). Alternativ kann ein Doppelstrangbruch aber auch durch ionisierende Strahlung oder Chemikalien ausgelöst werden. Dies wäre dann die Verbindung der homologen Rekombination mit der DNA-Reparatur.

Was wirklich während der homologen Rekombination passiert, wissen wir noch nicht hundertprozentig. Man kann diesen vorgang nicht live an einem DNA-Molekül mitverfolgen. Man kann aber seine Versuche so aufbauen, dass bestimmte Abläufe aufgrund des Ergebnisses ausgeschlossen werden können, andere Abläufe aber wahrscheinlicher werden. Viele solcher grundlegenden Experimente wurden mit der Bäckerhefe Saccharomyces cerevisiae gemacht, bei der clever definierte Konstrukte in das Genom eingebracht wurden, die nach einer homologen Rekombination erlauben, Anteile von bestimmten Vorgängen zu ermitteln. Das hört sich jetzt sehr nichtssagend an, deshalb will ich kurz an einem schönen Beispiel zeigen was ich damit meine.

Das "Double Strand Break Repair" Modell der homologen Rekombination
1983 fassten Szostak et al. in einem Review-Artikel den damals aktuellen Stand der Rekombinationsforschung zusammen. Dabei stellten sie fest, dass bisher vorgeschlagene Modelle der homologen Rekombination selten auftrende Ereignisse während der Meiose nicht vorhersagen konnten. Wenn man beispielsweise zwei Marker betrachtet [1], die heterozygot und gekoppelt vorliegen (also gemeinsam auf einem Chromosom sitzen, aber nicht auf dem zweiten Chromosom eines diploiden Organismus), dann erwartet man in den Nachkommen die einfache Aufspaltung nach Mendel in 1:1 - eine Hälfte der Nachkommen hat das Chromosom mit beiden Markern erhalten, die andere Hälfte das homologe Chromosom ohne die Marker. Aufgrund der besonderen Chromosomensituation der damals untersuchten Pilzarten ist die Notation hier traditionellerweise nicht 1:1, sondern 4:4, das ändert am Verhältnis aber nichts. Man kann nun aber beobachten, dass manchmal auch andere Aufteilungen passieren, beispielsweise 6:2 (bzw. 2:6), oder auch 5:3. Hier muss Information von einem Chromosom auf ein anderes übertragen worden sein, oder ein Austausch zwischen zwei Chromosomen erfolgt sein, so dass die beiden Marker nicht mehr gekoppelt auf einem Chromosom vorliegen und unabhängig voneinander vererbt werden können. Diese beiden Vorgänge bezeichnet man als gene conversion und crossing over , und sie sind in Abbildung 1 aus Szostak et al. dargestellt (wenn auch in umgekehrter Reihenfolge):

Abbildung 1 aus Szostak et al. (1983). Zustand von zwei gekoppelten heterozygoten Markern A und B und die Effekte von crossing over (a) und gene conversion (b). Klicken für größere Version.

Ausgehend von einem Doppelstrangbruch werden die freien Enden durch Proteine (Exonukleasen) so zurückgeschnitten, dass freie einzelsträngige Überhänge vorliegen. So eine ssDNA ist dann natürlich verfügbar für Basenpaarungen. Wenn nach einer "Homologiesuche" eine homologe DNA-Sequenz gefunden wird (die wie gesagt beispielsweise auf dem homologen Chromosom, oder auch dem Schwesterchromatid liegen kann), dann erfolgt die sogenannte Einzelstranginvasion: Der Einzelstrang lagert sich an die komplementäre Sequenz an und verdrängt dabei einen der vorhandenen Stränge. Die daraus resultierende Struktur wird D-Loop (displacement loop) genannt. Von hier an kann das freie Ende mit Hilfe einer DNA-Polymerase verlängert werden, was den D-Loop vergrößert. Irgendwann ist ein so großer Bereich DNA im D-Loop verdrängt, dass dieser mit dem zweiten freien Ende des Doppelstrangbruches paaren kann. Dies bedeutet auch, dass das erste freie Ende endlich mit der anderen Seite des Doppelstrangbruches verknüpft werden kann. Der DSB ist jetzt zwar repariert, aber wir haben nun eine problematische DNA-Struktur vorliegen - zwei DNA-Moleküle sind an zwei Positionen überkreuzt. So eine kreuzförmige DNA-Struktur nennt man übrigens nach ihrem Erstbeschreiber Holliday Junction (AHA!), bei den zwei Überkreuzungen hier spricht man von einer doppelten Holliday Junction (dHJ). Warum ist diese Struktur problematisch? Weil eine Zelle sich nicht teilen kann, bevor die dHJ aufgelöst wurde!
Und jetzt greift die Idee von Jack Szostak und Kollegen. Eine Endonuklease, also ein Protein das im Inneren eines DNA-Moleküles schneidet, kann an den Holliday Junctions Schnitte setzen, um die zwei Stränge voneinander zu trennen. Und abhängig davon, ob die beiden Schnitte symmetrisch (unten links) oder asymmetrisch (unten rechts) erfolgen, erhält man entweder ein crossing over (CO, rechts) oder eine gene conversion (auch noncrossover genannt, also NCO, links).

Seit 1983 ist viel Zeit vergangen, doch das DSBR-Modell hat sich gehalten. Mittlerweile wurde etwa gezeigt, dass in der Meiose ein spezielles Protein absichtlich Doppelstrangbrüche zur Einleitung der homologen Rekombination setzt: SPO11. Auch Holliday Junctions wurden bereits als Intermediate der Rekombination experimentell nachgewiesen.

"Synthesis-dependent strand-annealing" und das "revised model "
Um es jetzt noch ein wenig komplizierter zu machen, will ich der Vollständigkeit halber das Bild auf den aktuellen Stand bringen. Als 1994 die Gruppen von William Engels und Gregory Gloor (Nassif et al., 1994) Rekombinationsexperimente mit der Fruchtfliege Drosophila melanogaster machten, stießen sie auf Ergebnisse, die sich mit dem DSBR-Modell von Szostak et al. nicht vollständig erklären ließen. Letzen Endes waren ihre Ergebnisse nur zu verstehen, wenn man beiden freien Enden des DSB erlaubte, unabhängig voneinander die Rekombination mit verschiedenen Partnern einzuleiten. Eine Auflösung nach ihrem synthesis-dependent strand-annealing -Modell (SDSA) benötigte demnach keine Holliday Junction als Intermediat. Bereits nach Verlängerung des ersten freien Endes würde dieses aus dem D-Loop geworfen und für die Basenpaarung mit dem zweiten Ende zur Verfügung stehen. Durch solch einen Mechanismus wären keine Crossoverprodukte möglich.
2001 wurden die beiden konkurrierenden Modelle DSBR und SDSA dann gleichzeitig von zwei Gruppen zusammengefasst. In dem heute als revised model bezeichneten Prozess nach in der Bäckerhefe gewonnenen Ergebnissen von Hunter und Kleckner (2001) und Allers und Lichten (2001) beginnt die homologe Rekombination zunächst, wie ich es schon für DSBR und SDSA beschrieben habe. Die Aufteilung in die beiden Arme CO und NCO erfolgt jedoch schon vor dem dHJ-Intermediat, nämlich auf Ebene des D-Loop. Von hier ab kann dann die Rekombination entweder zum NCO aufgelöst werden, per SDSA-Modell. Oder eben über die doppelte Holliday Junction zum CO, das nach diesem Modell das einzige Ergebnis des DSBR-Weges ist.

revised model der homologen Rekombination nach Hunter und Kleckner (2001) und Allers und Lichten (2001). Klicken für größere Version.

Auf diesem Stand möchte ich es dann auch belassen für heute. Worauf ich hier überhaupt nicht eingegangen bin, sind die vielen Proteine, die diese ganzen Wege bevölkern. Von manchen kennt man recht gut ihre Position im Schema und ihre dortige Aufgabe, von vielen anderen weiß man aber nur ungefähr, in welche Hälfte des Modells sie passen.
Im nächsten richtigen Post dieser kurzen Serie werde ich dann, bewaffnet mit dem Hintergrund zur homologen Rekombination hier, Untersuchungen unserer Mutanten zur Rekombinationsrate vorstellen.

[1] Vor der Zeit der schnellen Sequenzierung, und auch vor dem Triumphzug der PCR waren solche Marker oft Sporenfarbgene der untersuchten Pilze, oder Antibiotika-Resistenzgene.

JW Szostak, TL Orr-Weaver, RJ Rothstein, FW Stahl (1983). The double-strand-break repair model for recombination Cell, 33 (1), 25-35 DOI: 10.1016/0092-8674(83)90331-8
N Nassif, J Penney, S Pal, WR Engels, GB Gloor (1994). Efficient copying of nonhomologous sequences from ectopic sites via P-element-induced gap repair. Mol Cell Biol., 14 (3), 1613-1625
Neil Hunter, Nancy Kleckner (2001). The Single-End InvasionAn Asymmetric Intermediate at the Double-Strand Break to Double-Holliday Junction Transition of Meiotic Recombination Cell, 106 (1), 59-70 DOI: 10.1016/S0092-8674(01)00430-5
T Allers, M Lichten (2001). Intermediates of Yeast Meiotic Recombination Contain Heteroduplex DNA Molecular Cell, 8 (1), 225-231 DOI: 10.1016/S1097-2765(01)00280-5

Crossing over : Holliday model - Biology

A number of models have been put forth over the years to explain how DNA recombination occurs. The basis of these models lies in what has been learned of recombination from yeast and other fungi. Fungi have been studied intensively because they possess certain properties such as the ability to yield four viable meiotic products (spores) that can be assayed and analyzed. For example, a mutation that is heterozygous will give rise to two mutant spores and two wild type spores, a 2+:2- pattern that is considered to be classically Mendelian. The appearance of non-Mendelian patterns such as 1+:3- (a "gene conversion") or the presence of "sectored" colonies (colonies with a divided phenotype) gave rise to a series of models to explain these events. While it is interesting how different models evolved, we concentrate here on a basic working model of DSB-induced recombination.

The diagram below shows a working model of how DNA double strand break induced recombination can occur.

More generally, a gene conversion refers to the unidirectional transfer of genetic information as opposed to a reciprocal exchange of information. In S. cerevisiae, gene conversion is recognized as a fundamental recombination process that can occur with or without crossing over of adjacent sequences.

Meiotic recombination
Experimental evidence for this model comes in the form of genetic and physical evidence. Physical analysis of DNA isolated from cells undergoing meiosis, for example, shows that DSBs are formed and they occur at recombination hotspots. For example, they occur in regions of high gene conversion that decreases with increasing distance from the DSB, a phenomenon termed polarity. Furthermore, these experiments also showed that DSBs lead to the formation of single stranded tails. Gel electrophoretic experiments have also provided evidence for structures that appear to be double Holliday structures. Gene conversion gradients are lost in mutants deficient in mismatch repair, indicating that gene conversion tracts are formed by long stretches of heteroduplex DNA. Special mutations that cannot be mismatch-corrected also indicate the presence of long heteroduplex tracts.

Mitotic recombination.

Break-induced recombination can also be studied in cells growing mitotically. In our lab we study this by inducing the HO endonuclease which initiates the process of mating type switching in yeast. The HO endonuclease cleaves the DNA sequence at MAT, the mating type locus, that determines the cell's mating type (either a or alpha). The DSB causes a gene conversion event to occur using either of two donor sequences, called HML and HMR, leading to the unidirectional transfer of mating type information from HML or HMR to MAT. Which donor is preferred depends on a sequence called the recombination enhancer which we consider in another part of this web site (Link to donor preference).


Crossing over results in the formation of new combination of characters in an organism called recombinants. In this, segments of DNA are broken and recombined to produce new combinations of alleles. This process is called Recombination. (Figure 3.12)

The widely accepted model of DNA recombination during crossing over is Holliday’s hybrid DNA model. It was first proposed by Robin Holliday in 1964. It involves several steps. (Figure 3.13)

1. Homologous DNA molecules are paired side by side with their duplicated copies of DNAs

2. One strand of both DNAs cut in one place by the enzyme endonuclease.

3. The cut strands cross and join the homologous strands forming the Holliday structure or Holliday junction.

4. The Holliday junction migrates away from the original site, a process called branch migration, as a result heteroduplex region is formed.

5. DNA strands may cut along through the vertical (V) line or horizontal (H) line.

6. The vertical cut will result in hetero duplexes with recombinants.

7. The horizontal cut will result in hetero duplex with non recombinants.

Calculation of Recombination Frequency (RF)

The percentage of recombinant progeny in a cross is called recombination frequency. The recombination frequency (cross over frequency) (RF) is calculated by using the following formula. The data is obtained from alleles in coupling configuration (Figure 3.14)

Content Review

Don’t spend a lot of time reviewing concepts at this point. The Guided Practice activity will serve as a more complete review later in the lesson. The animation serves primarily as an aid to help students visualize the crossing over process so that they will be better equipped to complete the hands-on modeling activity that will follow.

Share a brief animation, Meiosis: Crossing-Over.

Be prepared to stop the video clip at different points to ask questions:

Look for students to identify that sister means that the chromatids are a part of the same chromosome, with the same traits coded on each. Having seen the images of a dyad and tetrad in the video clip, students should also be able to explain that dyad means 1 pair of homologous chromosomes that result when a tetrad divides and tetrad means 2 homologous chromosomes.


Setting the Stage

To get students' attention, wonder and engage them in the introductory part of the course, the students are shown photographs of parents and children, and asked what characteristics children inherit from their fathers and mothers (Figure 1). The purpose of showing Figure 1 is to motivate students and understand the connection between homologous chromosomes and their similarity with their relatives. Questions for students with Figure 1 are: Are these people related or not? Why do you think they are relatives? Which characteristics of the boy resembles his father? Do you resemble your parents? Which characteristics of yours resemble to your parents? And finally: Why do you look like your parents? The students name some of the characteristics people receive from their parents.

Following this, the teacher asks students why children are not identical to their parents, to increase students' sense of wonder. By asking these questions, the teacher will help students to connect what they already know with the concept of homologous chromosomes and crossing over. Thus, students connection their daily life experiences with these concepts. By understanding these concepts, students comprehend the underlying information related to the resemblance between children and their parents. Thus, the teacher helps students to construct crossing over and daily life, using the correct model for elucidating this process.

Watch the video: Holliday Model of Recombination (May 2022).


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