1. Models for Homologous Recombination a. Key steps of homologous recombination i. Alignment of two homologous DNA molecules 1.

Homologous: identical or near identical for at least 100bp 2. Alleles: small regions of sequence and may carry different sequence variants ii. Introduction of breaks in the DNA iii. Formation of initial short regions of base pairing between the two recombining DNA molecules 1. Strand invasion occurs a. Forms a heteroduplex DNA i. Strands of DNA often containing mismatched base pairs, generated from strand invasion iv. Formation of a Holliday junction 1. Two DNA molecules that are connected by a crossing of the strands 2. The junction can move by branch migration a. Occurs when the function moves by melting and forming base pairs along the DNA v. Cleavage of the Holliday junction 1. Regenerates two separate duplex DNA molecules 2. Known as resolution b. Strand Invasion is a Key Step in Homologous Recombination i. Recombination is initiated by the presence of a double-stranded break in one of the DNA molecules ii. Strand invasion is the central step in homologous recombination iii. Strand invasion generates a Holliday junction that can then move along the DNA by branch migration 1. This migration increases the length of DNA exchanged iv. Repair of mismatches in the heteroduplex DNA can have important genetic consequences c. Resolving Holliday Junctions is a Key Step to Finishing Genetic Exchange i. Finishing recombination requires resolution of the Holliday junction by cutting the DNA strands near the site of the cross ii. Vertical cut sites of rotated Holliday junctions occur in the two DNA strands that are composed entirely of DNA from one of the two parental DNA molecules 1. Product are "spliced" a. Recombination products contain the two original duplexes and are now spliced together such that regions from the parental DNA molecules are covalently joined together by a region of hybrid duplex b. Also called crossover product iii. Horizontal cuts of rotated Holliday junctions occur in the two DN strands that contain regions of sequences from both paternal molecules 1. After resolution and covalent joining of the strands at these sties, the resulting DNA molecules contain a region of "patch" or hybrid DNA

a. Known as patch product 2. In these products, recombination does NOT result in reassortment of the genes flanking the site of the initial cleavage; these are known as noncrossover products d. The Double-Stranded Break-Repair Model Describes Many Recombination Events i. Since homologous recombination is initiated by a double-stranded break, the model describes the type of genetic exchange is the double-stranded breakrepair pathway 1. Starts with the introduction of a DSB in one of two homologous duplex DNA molecules ii. After introduction of the DSB, a DNA cleaving enzyme sequentially degrades the broken DNA molecule to generate regions of ssDNA 1. This process creates single-stranded extensions, known as ssDNA tails, on the broken DNA molecules these ssDNA tails terminate with 3' ends iii. The ssDNA tails then invade the unbroken homologous DNA duplex 1. The invading strand base-pairs with its complementary strand in the other DNA molecule 2. Invading strands end with 3' termini; therefore they are primers for new DNA synthesis a. Elongation from these DNA ends using complementary strands in the homologous duplex as a template serves to regenerate the regions of DNA that were destroyed during the process of the strands at the break site 2. Homologous Recombinatino Protein Machines a. RecBCD Pathway i. Is the E. coli major DSB-repair pathway ii. Requires a DSB on one of the recombining two DNA molecules 1. No protein has been found in Bacteria that induces DSBs 2. Breaks generated as a result of DNA damage or failure of a replication fork are the major sources of the initiating events b. RecBCD Helicase/Nuclease Processes Broken DNA Molecules for Recombination i. DNA molecules with ssDNA tails are the preferred substrate for initiating strand exchange between regions of homologous sequence ii. RecBCD helps load the RecA (strand exchange protein) onto the ssDNA ends iii. RecBCD is composed of three subunits: 1. RecB 2. RecC 3. RecD a. And has both DNA helicase and nuclease activity b. Binds to DNA molecules at the site of a DSB and tracks along DNA using the energy of ATP hydrolysis i. As a result, the DNA is unwound with or without the accompanying nucleolytic destruction of one or both strands c. The activities of RecBCD are controlled by specific DNA sequence elements known as Chi Sites (Crossover Hotspot Instigator) iv. Mechanism:

v.

1. RecBCD enters the DNA at the site of the DSB and moves along the DNA, unwinding the strands 2. RecB and RecD subunits are both DNA helicases a. RecB is a 3'-->5' helicase; slow and has a multifunctional DNA nuclease domain b. RecD is a 5'-->3' helicase; processes faster than RecB 3. RecC functions to recognize the Chi sites a. Upon encountering the Chi sequence, the nuclease activity of RecBCD is altered i. RecBCD moves into the sequence distal to the Chi site and no longer cleaves the DNA strand with 3'-->5' polarity ii. After the encounter, the other DNA strand (5'-->3' polarity) is cleaved even more frequently than prior to the Chi site 1. As a result of this change, a duplex DNA molecule is converted into one with a 3' single strand extension terminating with the Chi sequence at the 3' end 1. Structure is ideal for assemble of RecA and initiation of stand exchange 2. Molecular basis of the change in RecBCD enzyme activity after the encounter with a Chi site is due to inactivator of the RecD subunit and a change in the way the DNA travels through the multisubunit RecBCD complex Structure of RecBCD 1. Protein complex has an overall triangular shape, with the Duple DNA entering the protein from the top point of the triangle a. Here, the DNA encounters a "pin" structure protruding from the RecC subunit that splits the duplex and guides the two individual strands of DNA to the two motors within the enzyme i. The RecC subunit channels the 3' strand to the RecB motor and the 5' strand do the RecD motor 1. RecC, although not a helicase, contributes to the overall efficiency of the helicase activity of the complex ii. The organization of the channels within the enzyme causes the 3' tail to be fed along a groove that emerges at the nuclease active site on the RecB subunit 1. Results in the strand is now effectively and processively degraded, prior to the enzyme encountering a Chi site iii. The 5' tail also moves by the nuclease active site upon leaving the RecD motor, but it is digested less frequently than the 3' trail because it must compete with the more favorable positioned 3' strand b. Upon entering the Chi site, the situation changes i. RecC recognizes and binds tightly to this DNA site, and once the 3' end is bound, it is prevented from entering the nuclease

1. Binding both prevents further digestion of the 3' tail and promotes digestion of the 5' tail by removing its competitor ii. The ssDNA tail generated by RecBCD must be coated by the RecA protein before recombination can occur 1. Cells also contain ssDNA binding proteins that can bind to the DNA 1. To ensure that RecA and NOT SSBs bind to DNA, RecBCD interacts directly with RecA and promotes its assembly 1. Loading activity involves a direct proteinprotein interaction between the nuclease domain of the RecB subunit and the RecA protein and serves to load RecA on the DNA with the 3' tail c. Chi Sites Control RecBCD i. Chi sites increase frequency of recombination approximately 10 fold 1. Elevated recombination frequencies are observed for approximately 10kb distal to the chi site, and drop off gradually over a distance a. This is because the DNA between the DSB (where RecBCD enters) and the chi site is cut into small pieces by the enzyme and is therefore not available for recombination ii. The ability of chi sites to control the nuclease activity of RecBCD also helps bacterial cells protect themselves from foreign DNA that may enter via phage infection or conjugation 1. Eight-nucleotide chi site (GCTGGTGG) is highly overrepresented in the E. coli genome which occurs about 80 times which is equivalent to approximately 1009 chi sites 2. The E. coli DNA that enters an E. coli cell is likely to be processed by RecBCD in a manner that generates the 3' ssDNA tails, and thus to be activated for recombination a. DNA from another species will lack frequent chi sites i. RecBCD action on this DNA will lead to its extensive degradation, rather than activation for recombination iii. The DNA-degradation activity of RecBCD has multiple consequences: 1. Needed to process DNA at a break site for the subsequent steps of RecA assembly and strand invasion a. In this manner, RecBCD promotes recombination b. Because RecBCD degrades DNA to activate it, the overall process of homologous recombination must also involve DNA synthesis to regenerated the degraded strands 2. Functions simply to destroy DNA as it does when foreign DNA lacking frequent chi sites enters cell a. Protects cells from the potentially deleterious consequences of taking up foreign sequences d. RecA Protein Assembles on Single-Stranded DNA and Promotes Strand Invasion

RecA is the central protein in homologous recombination 1. Founder member of a family of enzymes called strand-exchange protein a. Catalyze the pairing of homologous DNA molecules i. Pairing involves both the search for sequence matches between the two molecules and the generation of regions of base pairing between these molecules ii. The important features of the DNA molecules are: 1. DNA sequencing complementarity between the two partner molecules 2. A region of ssDNA on at least one molecule to allow RecA assembly 3. Presence of a DNA end within the region of complementarity, enabling the DNA strands in the newly formed duplex to intertwine iii. The active form of RecA is a protein-DNA filament 1. Filament is huge and variable in size; filaments that contain approximately 100 subunits of RecA and 300 nucleotides of DNA are common a. The filament can accommodate one, two, three, or four strands of DNA iv. The structure of DNA within the filament is highly extended compared to either uncoated ssDNA or a standard B-form helix 1. The distance between adjacent bases is 5 , in contrast to the 3.4 spacing 2. Upon RecA binding to the DNA molecule, it will be extended by approximately 1.5 fold v. To form a filament, subunits of RecA bind cooperatively to DNA 1. RecA binding and assembly are much more rapid on ssDNA than on dsDNA a. Filament grows by the addition of RecA subunits in the 5'-->3' direction, such that a DNA strand that reminates in 3' ends is most likely to be coated by RecA e. Newly Base-Paired Partners are Established Within the RecA Filament i. RecA-catalyzed strand exchange can be divided into distinct reaction stages: 1. RecA filament must assemble on one of the participating DNA molecules a. Assembly occurs on a molecule containing a region of ssDNA (ssDNA tail) b. This RecA-ssDNA complex is the active form that participates in the search for a homology 2. Search for homology a. RecA must look for base-pair complementarity between the DNA within the filament and a new DNA molecule b. This homology search is promoted by RecA because the filament structure has two distinct DNA binding sites: i. Primary site 1. Bound by the first DNA molecule ii. Secondary site 1. Can be occupied by a double-stranded DNA 2. Binding is rapid, weak, transient, and independent of DNA sequence c. How does the RecA filament sense homology?

i.

Not yet clear DNA in the secondary binding site is transiently opened and tested for complementarity with the ssDNA in the primary site 1. This testing is presumable via base pairing interactions 2. Experiments suggest that the initial alignment may involve base flipping of some of the bases in the DNA duplex 3. In vitro experiments indicate that a sequence match of just 15bp provides a sufficient signal to the RecA filament that a match has been found 3. Once a region of base-pair complementarity has been located, RecA promotes the formation of a stable complex between these two DNA molecules a. The RecA-bound three stranded structure is called a joint molecule and usually contains several hundred base pairs of hybrid DNA i. It is within the joint molecule that the actual exchange of DNA strands occurs 1. The DNA strand in the primary binding site becomes base paired with its complement in the DNA duplex bound in the secondary site 2. Strand exchange require the breaking of one set of base pairs and the formation of a new set of identical base pairs b. RecA binds preferentially to the DNA product after strand exchange has occurred, and it is this binding energy that actually drives the exchange reaction toward the new DNA configuration f. RecA Homologs are Present in All Organisms i. Strand-exchange proteins of the RecA family are present in all forms of life 1. Best-characterized list: a. RecA in Eubacteria b. RadA in Archaea c. Rad51 and Dmc1 in Eukaryota d. UvsX protein in Bacteriophage T4 g. The RuvAB Complex Specifically Recognizes Holliday Junctions and Promotes Branch Migration i. After the strand invasion step of recombination is complete, the two recombining DNA molecules are connected by a DNA branch known as a Holliday junction 1. Movement of the site of this branch requires exchange of DNA base pairs between the two homologous DNA duplexes ii. RuvA protein is the Holliday junction-specific DNA-binding protein that recognizes the structure of the DNA junction, regardless of its specific DNA sequence 1. Recognizes and binds Holliday junctions and recruits the RuvB protein to this site

i. ii.

RuvB is a hexameric ATPase, similar to the hexameric helicases involved in DNA replication 1. Provides the energy to drive the exchange of base pairs that move the DNA branch a. This energy is needed to move the branch rapidly and in one direction iv. Structural models of RuvAB complexes at a Holliday junction show how a tetramer of RuvA together with two hexamers of RuvB work together to power this DNA-exchange process h. RuvC Cleaves Specific DNA Strands at the Holliday Junction to Finish Recombination i. Completion of recombination requires that the Holliday junction between the two recombining DNA molecules be resolved 1. In bacteria, the major Holliday junction resolving endonuclease is RuvC a. Discovered and purified based on its ability to cut DNA junctions made by RecA in vitro ii. Resolution by RuvC occurs when RuvC recognizes the Holliday junction-likely in a complex the RuvA and RuvB-and specifically nicks two of the homologous DNA strands that have the same polarity 1. Cleavage results in DNA ends that terminate with 5'-phosphates and 3'-OH groups that can be directly joined by DNA ligase iii. RuvC cleaves DNA with modest sequence specificity 1. Cleavage takes place only at sites conforming to the consensus 5'A/T-T-TG/C-3' a. Cleavage occurs after the second T in the sequence; this sequence is found frequently in DNA averaging one every 64 nucleotides i. This ensures at least some branch migration occurs before resolution 3. Homologous Recombination in Eukaryotes a. Homologous Recombination has Additional Functions in Eukaryotes i. Homologous recombination in bacteria is required to: 1. Repair DBSs in DNA 2. Restart collapsed replication forks 3. Allow a cell's chromosomal DNA to recombine with DNA that enters via phage infection of conjugation ii. Homologous recombination is also required for DNA repair and the restarting of collapsed replication forks in eukaryotic cells iii. Animals with mutations that interfere with homologous recombination are predisposed to certain types of cancer iv. In eukaryotes, homologous recombination is required for proper chromosome pairing and thus for maintaining the integrity of the genome in meiosis b. Homologous Recombination is Required for Chromosome Segregation During Meiosis i. Before division, the cell has two copies of each chromosome, one each that was inherited from its two parents and during S phase, the chromosomes are replicated to give a total DNA content of 4N

iii.

In preparation of the first nuclear division, these duplicated homologous chromosomes must pair and align at the center of the cell iii. Recombination must be completed before the first nuclear division to allow the homologs to properly align and then separate 1. During this process, the sister chromatids remain paired 2. In the second nuclear division, it is the sister chromatids that separate iv. In the absence of recombination, chromosomes often fail to align properly for the first meiotic division, and as a result, there is a high incidence of chromosome loss 1. This improper segregation of chromosomes (nondisjunction) leads to a large number of gametes without the correct chromosome complement v. The homologous recombination events that occur during meiosis are called meiotic recombination 1. Frequently gives rise to crossing over between genes on the two homologous parental chromosomes a. The genetic exchange, can be observed cytologically c. Programmed Generation of Double-Stranded DNA Breaks Occurs During Meiosis i. The developmental program needed for cells to successfully complete meiosis involves turning on the expression of many genes that are NOT needed during normal growth 1. SPO11 a. Encodes a protein that introduces DBSs in chromosomal DNA to initiate meiotic recombination b. Protein cuts the DNA at many chromosomal locations, with little sequence selectivity, but at a very specific time during meiosis c. Spo-11 mediated DNA cleavage occurs right around the time when the replicated homologous chromosomes start to repair i. Spo11 cut sites are not randomly distributed along the DNA; rather are located most commonly in chromosomal regions that are not tightly packed with nucleosomes, such as promoters controlling gene transcription d. Mechanism: i. Specific tyrosine side chain in the Spo11 protein attacks the phosphodiester backbone to cut the DNA and generate a covalent complex between the protein and the severed DNA strand 1. Two subunits of Spo11 cleave the DNA two nucleotides apart on the two DNA strands to make a staggered DSB 1. Spo11 shares this DNA cleavage mechanism with the DNA topoisomerases and the site-specific recombinases 2. The fact that Spo11 cleavage involves a covalent proteinDNA complex has two consequences: 1. The 5' ends of the DNA at the site of Spo11 cleavage are covalently bound to the enzyme

ii.

1. These Spo11-linked 5'-DNA ends that are the initial sites of DNA processing to create the ssDNA tails required for assembly of RecAlike proteins and initiation of strand invasion ii. The energy of the cleaved DNA phosphodiester bond is stored in the bound protein-DNA linkage, and so the DNA strands can be resealed by a simple reversal of the cleavage reaction 1. Resealing can occur when cells receive a signal to stop proceeding with meiosis d. MRX Protein Processes the Cleaved DNA Ends for Assembly of the RecA-Like Strand-Exchange Proteins i. During meiotic recombination, the MRX-enzyme is responsible for this DNAprocessing event 1. This complex (although NOT homologous to RecBCD) is a multisubunit DNA nuclease 2. Composed of a protein subunits called: a. Mre11 b. Rad50 c. Xrs2 ii. Processing of the DNA at the break site occurs exclusively on the DNA strand that terminates with a 5' end (the strands covalently attached to the Spo11 protein) 1. The strands terminating with 3' ends are NOT degraded a. This reaction is therefore called a 5'-->3' resection i. Generates the long ssDNA tails with 3' ends that are often 1kb or longer iii. The MRX complex is thought to remove the DNA-linked Spo11 e. Dmc1 Is a RecA-Like Protein That Specifically Functions in Meiotic Recombination i. Eukaryotes encode two well-characterized homologs of the bacterial RecA protein: 1. Rad51 a. Widely expressed in cells dividing mitotically and meiotically 2. Dmc1 a. Expressed only as cells enter meiosis i. Both proteins function in meiotic recombination ii. Strand exchange during meiosis occurs between a particular type of homologous DNA partner 1. Recall: meiotic recombination occurs at a time when there are four complete, dsDNA molecules and the two homologs each of which have been copied to generate two sister chromatids a. Although the two homologs likely contain small sequence differences and carry distinct alleles for various genes, the majority of the DNA sequence among these four copies of the chromosome will be identical

Dmc1-dependent recombination is preferentially between the nonsister homologous chromatids rather than between the sisters 1. Rationale: meiotic recombination promotes interhomolog connections to assist alignment of the chromosomes for division f. Many Proteins Function Together to Promote Meiotic Recombination i. Protein involved in the critical stages of DSB formation, DNA processing to generate 3' ssDNA tails, and strange exchange during meiotic recombination have been identified and characterized ii. Many proteins appear to interact with the known recombination enzymes,, and it seems likely that these proteins function in the context of a large multicomponent complex 1. These large protein-DNA complexes are known as recombination factories and can be visualized in cells iii. Various other proteins have been shown to be involved with Rad51 to help promote recombination and DSB repair 1. Rad52 is another essential recombination protein that interacts with Rad51 a. Functions to promote assembly of Rad51 DNA filaments, the active form of Rad51 i. It accomplishes this by antagonizing the action of RPA, the major ssDNA-binding protein present in eukaryotic cells 1. In hindsight, Rad52 shares an activity with the E. coli RecBCD protein which helps RecA load onto ssDNA that would otherwise have been bound to SSB ii. Rad52 protein also promotes the annealing and base pairing of complementary ssDNA molecules 2. The product of the BRCA2 gene also participates in Rad51-mediated DSB repair iv. By analogy with Bacteria, we expect that eukaryotic cells encode proteins that promote the branch migration and Holliday junction resolution steps of recombination 1. In fact, enzymes promoting these reactions are being identified a. Complex containing a Rad51-like protein called Rad51C and XRCC3 which both have been found to contain Holliday junction resolvase activity 4. Mating-Type Switch a. In addition to promoting DNA pairing, DNA repair, and genetic exchange, homologous recombination also serves to change the DNA sequence at a specific chromosomal location i. Often used to regulate gene expression b. S. cerevisiae is a single-cell eukaryote that can exist as any of three different cell types: i. Haploid Sc cells can be either of two mating types: 1. a 2.

i.

a. In addition, when an a or cell come into close proximity, they can fuse to form an a/ diploid cell i. The cell may then go through meiosis to form two haploid a cells and two cells c. The mating type genes encoding transcriptional regulators i. Regulators control expression of target genes whose products define each cell type ii. Mating-type genes expressed in a given cell are those found at the mating type locus (MAT locus) in that cell 1. This in a cells the a1 gene is present at the MAT locus, whereas in cells, the 1 and 2 genes are present at the MAT locus d. Cells can switch their mating type by recombination i. In addition to the a or present at the MAT locus in each cell, there is an additional copy of both the a and genes present (but NOT expressed) elsewhere in the genome 1. These silent copies are found at loci called HMR and HML a. Known as silent cassettes b. Their function is to provide a storehouse of genetic information that can be used to switch a cells' mating type c. The switch requires the transfer of genetic information from the HM sites to the MAT locus via homologous recombination e. Mating-Type Switching is Initiated by a Site-Specific Double-Strand Break i. Mating-type switching is initiated by the introduction of the DSB at the MAT locus 1. This reaction is performed by a specialized DNA-cleaving enzyme called HO endonuclease a. Expression of the HO gene is tightly regulated to ensure that switching occurs only when it should b. Sequence-specific endonuclease; the only sites in the yeast chromosome that carry HO recognition sequences are the mating type loci c. HO cutting introduces a staggered break in the chromosome; and in contrast to Spo11 cleavage, HO simply hydrolyzes the DNA and does not remain covalently linked to the cut strand ii. 5'-->3' resection of the DNA at the site of the HO-induced break occurs by the same mechanism used during meiotic recombination 1. Resection depends on the MRX protein complex and is specific for the strands that terminate with 5' ends 2. In contrast, the 3' terminating ends are very stable and not subjected to nuclease digestion a. Once the 3' ssDNA tails have been generated, the DNA becomes coated by the Rad51 and Rad52 proteins i. These Rad51 protein-coated strands then search for homologous chromosomal regions to initiate strand invasion and genetic exchange

Mating-type switching is unidirectional; sequence information is "moved" from the MAT locus, from HMR and HML, but information never "goes" in the other direction 1. The cut MAT locus is always the recipient partner during recombination, and the HMR and HML sites remain unchanged by the recombination process a. This stems from the fact that HO endonuclease cannot cleave its recognition sequence at either HML or HMR because the chromatin structure renders these sites inaccessible for this enzyme iv. The Rad51-coated 3'-ssDNA tails from the MAT locus choose the DNA at either the HMR or HML locus for strand invasion 1. If the DNA sequence at MAT is a, then invasion occurs with HML, which carries the "storage" copy of the sequences 2. If the DNA sequence at MAT is , the invasion occurs with HMR, which carries the storage copy of the a sequences a. After recombination, the genetic information that was at the chosen HM loci is present at the MAT loci as well i. This genetic change occurs without a reciprocal swap of information from MAT to the HR locus f. Mating-Type Switching is a Gene Conversion Event and Not Associated with Crossing Over i. Evidence indicates that after the strand invasion step, this recombination pathway diverges from the DSB-repair mechanism 1. One hint that the mechanism is distinct is that the crossover class of recombination products is never observed during mating-type switch 2. Recall: in the DSB-repair pathway, resolution of the Holliday junction intermediates gives two classes of products: the splice (crossover) or the patch (noncrossover) a. Theories state that these resolutions occur at equal frequencies; however, crossover products are rarely observed in mating-type switching ii. Gene conversion without crossing over, a new recombination model termed synthesis-dependent strand annealing (SDSA) 1. The initiating even is the introduction of a DSB at the recombination site 2. After strand discharge, the invading 3' end serves as the primer to initiate new DNA synthesis a. In contrast to what occurs during the DSB-repair pathway, a complete replication fork is assembled at this site 3. Both leading-strand and lagging-strand DNA synthesis occurs a. In contrast to normal DNA replication, the newly synthesized strands are displaced from the template 4. As a result, a new double-stranded DNA segment is synthesized, joined to the DNA site that was originally cut by HO, and resected by MRX 5. Completing recombination requires that the other "old" DNA strand present at MAT (the 3'-ending strand not cleaved by MRX) be removed

iii.

a. The newly synthesized DNA-an exact copy of the information in the partner DNA molecule-replaces the information that was originally present 5. Genetic Consequences of the Mechanism of Homologous Recombination a. A central feature of homologous recombination is that it can occur between any two regions of DNA, regardless of the sequence, provided these regions are sufficiently similar i. NONE of the steps in homologous recombination require recognition of a highly specific DNA sequence b. Distortions in genetic maps compared to physical maps occur when a region of DNA does not have the average probability of participating in recombination i. Regions with a higher than average probability are "hot spots"; whereas regions that participate less commonly than average segment are "cold spots" 1. Therefore two genes that have a hot spot between them appear in a genetic map to be farther apart than is true in a physical map of the same region 2. In contrast, genes separated by a "cold" interval appear by genetic mapping to be closer together than is true from their physical distance c. Encounter two examples of hot and cold spots i. Regions near chi spots and Spo11 cleavage sites have a higher than average probability of initiating recombination; whereas regions having few such sites are correspondingly "cold" d. One Cause of Gene Conversion is DNA Repair During Recombination i. Gene conversion is commonly observed during normal homologous recombination events, such as those responsible for genetic exchange in bacteria and for pairing chromosomes during meiosis (mating-type switching in yeast) ii. Illustration of Gene Conversion: 1. Consider a cell undergoing meiosis has the A allele on one homolog and the a allele on the other a. After DNA replication, four copies of this gene are present and the genotype would be A, A, a, a. b. In the absence of gene conversion, two gametes carrying the A allele and two gametes carrying the a allele would be generated i. If instead the gametes with genotypes A, a, a, a or A, A, A, a are formed, then a gene conversion even has occurred, in which one copy of the A gene has been converted into a (or vice versa). ii. How does this arise? iii. Two ways that gene conversion can occur during the DSB-repair pathway 1. Consider what could happen if the A gene was very close to the site of the DSB a. In this case, when the 3' ssDNA tails invade the homologous duplexes and are elongated, they may copy the a information, which could replace the A information in the product chromosome upon completion of recombination 2. Involves the repair of base-pair mismatches that occur in the recombination intermediates

a. If either strand invasion or branch migration includes the A/a gene, a segment of heteroduplex DNA carrying the A sequence on one strand would be formed i. This region of DNA carrying base-pair mismatches could be recognized and acted upon by the cellular mismatch repair enzymes ii. These enzymes are specialized for fixing base-pair mismatches in DNA 1. When they detect a mismatched base pair, these enzymes exist a short stretch of DNA from one of the two strands 2. A repair DNA polymerase then fills in the gap now with the properly base-paired sequence iii. When working o recombination intermediates, the mismatch repair enzymes will likely choose randomly which strand will repair 1. Therefore, after their action, both strands will carry the sequence coding either the A information or the a information (depending on which strand was "fixed" by the repair enzymes), and gene conversion will be observed