DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

The formation of heteroduplex DNA is a central step in the exchange of DNA sequences via homologous recombination, and in the accurate repair of broken chromosomes via homology-directed repair pathways. In cells, heteroduplex DNA largely arises through the activities of recombination proteins that promote DNA-pairing and annealing reactions. Classes of proteins involved in pairing and annealing include RecA-family DNA-pairing proteins, single-stranded DNA (ssDNA)-binding proteins, recombination mediator proteins, annealing proteins, and nucleases. This review explores the properties of these pairing and annealing proteins, and highlights their roles in complex recombination processes including the double Holliday junction (DhJ) formation, synthesis-dependent strand annealing, and single-strand annealing pathways—DNA transactions that are critical both for genome stability in individual organisms and for the evolution of species.A central step in the process of homologous recombination is the formation of heteroduplex DNA. In this article, heteroduplex DNA is defined as double-stranded DNA that arose from recombination, in which the two strands are derived from different parental DNA molecules or regions. The two strands of the heteroduplex may be fully complementary in sequence, or may contain small regions of noncomplementarity embedded within their otherwise complementary sequences. In either case, Watson-Crick base pairs must stabilize the heteroduplex to the extent that it can exist as free DNA following the dissociation of the recombination proteins that promoted its formation.The ability to form heteroduplex DNA using strands from two different parental DNA molecules lies at the heart of fundamental biological processes that control genome stability in individual organisms, inheritance of genetic information by their progeny, and genetic diversity within the resulting populations (Amunugama and Fishel 2012). During meiosis, the formation of heteroduplex DNA facilitates crossing-over and allelic exchange between homologous chromosomes; this process ensures that progeny are not identical clones of their parents and that sexual reproduction between individuals will result in a genetically diverse population (see Lam and Keeney 2015; Zickler and Kleckner 2015). Heteroduplex DNA generated by meiotic COs also ensures proper segregation of homologous chromosomes, so that each gamete receives a complete but genetically distinct set of chromosomes (Bascom-Slack et al. 1997; Gerton and Hawley 2005). In mitotic cells, heteroduplex DNA formation between sister chromatids is essential for homology-directed repair (HR) of DNA double-strand breaks (DSBs), stalled replication forks, and other lesions (Maher et al. 2011; Amunugama and Fishel 2012; Mehta and Haber 2014). Prokaryotic organisms also generate heteroduplex DNA to perform HR transactions, and to promote genetic exchanges, such as occur during bacterial conjugation (Cox 1999; Thomas and Nielsen 2005).Fundamentally, heteroduplex DNA generation involves the formation of tracts of Watson-Crick base pairs between strands of DNA derived from two different progenitor (parental) DNA molecules. Mechanistically, the DNA transactions giving rise to heteroduplex may involve two, three, or four strands of DNA (Fig. 1). DNA annealing refers to heteroduplex formation from two complementary (or nearly complementary) molecules or regions of single-stranded DNA (ssDNA) (Fig. 1A). DNA annealing may occur spontaneously, but it is promoted in vivo by certain classes of annealing proteins. Three-stranded reactions yielding heteroduplex DNA proceed by a different mechanism referred to as DNA pairing, strand invasion, or strand exchange. These reactions involve the invasion of a duplex DNA molecule by homologous (or nearly homologous) ssDNA. The invading DNA may be completely single stranded, as is often the case in in vitro assays for DNA-pairing activity (Fig. 1B) (Cox and Lehman 1981). Under physiological conditions, however, the invading ssDNA is contained as a single-stranded tail or gap within a duplex (Fig. 1C,D). DNA-pairing reactions are promoted by DNA-pairing proteins of the RecA family (Bianco et al. 1998), and proceed via the formation of D-loop or joint molecule intermediates that contain the heteroduplex DNA (Fig. 1B–D). Three-stranded reactions may also be promoted by exonuclease/annealing protein complexes found in certain viruses. Four-stranded reactions generating heteroduplex DNA involve branch migration of a Holliday junction (Fig. 1D). In practice, a four-stranded reaction must be initiated by a three-stranded pairing reaction catalyzed by a DNA-pairing protein, after which the heteroduplex is extended into duplex regions through the action of the DNA-pairing protein or of an associated DNA helicase/translocase (Das Gupta et al. 1981; Kim et al. 1992; Tsaneva et al. 1992).Open in a separate windowFigure 1.Common DNA annealing and pairing reactions. (A) Simple annealing between two complementary molecules of single-stranded DNA to form a heteroduplex. (B) Three-stranded DNA-pairing reaction of the type used for in vitro assays of RecA-family DNA-pairing proteins. The single-stranded circle is homologous to the linear duplex. Formation of heteroduplex (red strand base-paired to black) requires protein-promoted invasion of the duplex by the ssDNA to form a joint molecule or D-loop (i). The length of the heteroduplex may be extended by branch migration (ii). (C) Three-stranded DNA-pairing reaction of the type used for high-fidelity repair of DNA DSBs in vivo. The invading strand is the ssDNA tail of a resected DSB. The 3′ end of the invading strand is incorporated into the heteroduplex within the D-loop intermediate. (D) Example of a four-stranded DNA-pairing transaction that is initiated by a three-stranded pairing event and extended by branch migration. The ssDNA in a gapped duplex serves as the invading strand to generate a joint molecule (i), reminiscent of the reaction shown in panel B. Protein-directed branch migration may proceed into the duplex region adjacent to the original gap, generating α-structure intermediates (ii), or eventually a complete exchange of strands (iii).     

Heteroduplex joint formation by a stoichiometric complex of Rad51 and Rad52 of Saccharomyces cerevisiae

Both Rad51 and Rad52 are required for homologous genetic recombination in Saccharomyces cerevisiae. Rad51 promotes heteroduplex joint formation, a general step in homologous recombination. Rad52 facilitates the binding of Rad51 to replication protein A (RPA)-coated single-stranded DNA. The requirement of RPA can be avoided in vitro, if the single-stranded DNA is short. Using short single-stranded DNA and homologous double-stranded DNA, in the absence of RPA, we found that Rad52 (optimal at three per Rad51) was still required for Rad51-promoted heteroduplex joint formation in vitro, as assayed by the formation of D-loops, suggesting another role for Rad52. Rad51 has to bind to the single-stranded DNA before the addition of double-stranded DNA for efficient D-loop formation. Immunoprecipitation and single-stranded DNA-bead precipitation analyses revealed the presence of the free and DNA-bound complexes of Rad51 and Rad52 at a 1 to 2 stoichiometry. In the presence of single-stranded DNA, in addition to Rad51, Rad52 was required for extensive untwisting that is an intermediate step toward D-loop formation. Thus, these results suggest that the formation of the stoichiometric complex of Rad52 with Rad51 on single-stranded DNA is required for the functional binding of the protein-single-stranded DNA complex to the double-stranded DNA to form D-loops.     

Single-stranded conjugation in Escherichia coli K-12

Summary The mutation BT43 in the gene dnaB leads to the inhibition of vegetative and conjugational DNA synthesis at 42°. The consequences in case of conjugation are very unusual. The fragment of donor DNA tramsmitted to the recipient cell remains single-stranded and is integrated as such into the recipient chromosome similar to the main events during transformation. We call this process single-stranded (SS) conjugation.The evidence for this statement comes from the measurement of the time of expression of the gene tsx, containing the genetic information for the receptor of phage T6. The gene tsx is introduced into a dnaBT43 recipient cell alternatively by two different donors Hfr H and Hfr C, which are characterized by opposite directions of transfer. Therefore both donors introduce into the recipient cell alternatively the informational or noninformational DNA strand. If conjugation is performed at a nonpermissive temperature, the transferred DNA piece remains single-stranded and is integrated as such into the recipient chromosome. If it is the informational strand (case of Hfr H), it is transcribed very fast and yields the protein in question in about 20 min. If the noninformational strand is integrated (Hfr C) about 40 min additional time is required to effect cell division.SS-conjugation is very sensitive to the action of exonucleases Exo I and Exo V and is much enhanced in the absence of both nucleases in the recipient.The exogenous DNA pieces are integrated as short insertions, this leads to the disjoining of linked markers and to a very short scale of the genetic map. Because the donor DNA undergoes recombination in the single-stranded state heteroduplex regions originate which are subsequently corrected by the enzymes of the recipient cell. The situation leads to a very special but predictable heterogeneity of the progeny of transconjugants.The fact of the existence of this special process, SS-conjugation, drastically different from common conjugation in many respects, suggests that common conjugation leads to the integration of double-stranded DNA pieces into the recipient chromosome.     

Single-stranded DNA as a recombination substrate in plants as assessed by stable and transient recombination assays.

Two separate assays, one that requires stable integration of recombination products and one that does not, were employed to elucidate the role of single-stranded DNA in extrachromosomal homologous recombination in Nicotiana tabacum. Both assays revealed that single-stranded DNA in linear and in circular forms was an efficient substrate for recombination, provided that the cotransformed recombination substrates were of complementary sequence, so that direct annealing was possible. Recombination was inefficient when both single-stranded recombination partners contained homologous regions of identical sequence and generation of a double-stranded DNA was required prior to heteroduplex formation. These results indicate that direct annealing of single strands is an important initial step for intermolecular recombination in tobacco cells. Annealed cotransformed single-stranded molecules yielded intermediates that could be further processed by either continuous or discontinuous second-strand synthesis. The type of intermediate had no influence on the recombination efficiency. Double-stranded circles were unable to recombine efficiently either with each other or with single-stranded DNA. Our results suggest that a helicase activity is involved in the initial steps of double-stranded DNA recombination which unwinds duplex molecules at the site of double-strand breaks.     

The E.coli RuvAB proteins branch migrate Holliday junctions through heterologous DNA sequences in a reaction facilitated by SSB.

During genetic recombination a heteroduplex joint is formed between two homologous DNA molecules. The heteroduplex joint plays an important role in recombination since it accommodates sequence heterogeneities (mismatches, insertions or deletions) that lead to genetic variation. Two Escherichia coli proteins, RuvA and RuvB, promote the formation of heteroduplex DNA by catalysing the branch migration of crossovers, or Holliday junctions, which link recombining chromosomes. We show that RuvA and RuvB can promote branch migration through 1800 bp of heterologous DNA, in a reaction facilitated by the presence of E.coli single-stranded DNA binding (SSB) protein. Reaction intermediates, containing unpaired heteroduplex regions bound by SSB, were directly visualized by electron microscopy. In the absence of SSB, or when SSB was replaced by a single-strand binding protein from bacteriophage T4 (gene 32 protein), only limited heterologous branch migration was observed. These results show that the RuvAB proteins, which are induced as part of the SOS response to DNA damage, allow genetic recombination and the recombinational repair of DNA to occur in the presence of extensive lengths of heterology.     

Formation of heteroduplex DNA promoted by the combined activities of Escherichia coli recA and recBCD proteins

We have established an in vitro reaction in which heteroduplex DNA formation is dependent on the concerted actions of recA and recBCD proteins, the major components of the recBCD pathway of genetic recombination in vivo. We find that heteroduplex DNA formation requires three distinct enzymatic functions: first, the helicase activity of recBCD enzyme initiates heteroduplex DNA formation by unwinding the linear double-stranded DNA molecule to transiently form single-stranded DNA (ssDNA); second, recA protein traps this ssDNA before it reanneals; third, recA protein catalyzes the pairing of this ssDNA molecule with another homologous ssDNA molecule, followed by the renaturation of these molecules to form heteroduplex DNA. The first two functions should be important to all in vitro reactions involving recA and recBCD proteins, whereas the third will be specific to the DNA substrates used. The rate-limiting step of heteroduplex DNA formation is the trapping by recA protein of the ssDNA produced by recBCD enzyme. A model for this reaction is described.     

Saccharomyces cerevisiae Mer3 helicase stimulates 3'-5' heteroduplex extension by Rad51; implications for crossover control in meiotic recombination

Crossover and noncrossover recombinants can form by two different pathways during meiotic recombination in Saccharomyces cerevisiae. The MER3 gene is known to affect selectively crossover, but not noncrossover, recombination. The Mer3 protein is a DNA helicase that unwinds duplex DNA in the 3' to 5' direction. To define the underlying molecular steps of meiotic recombination, we investigated the role of Mer3 helicase in DNA strand exchange promoted by Rad51 protein. We found that Mer3 helicase does not function as an initiator of DNA pairing events but, rather, it stimulates DNA heteroduplex extension in the 3' --> 5' direction relative to the incoming (or displaced) single-stranded DNA. Conversely, Mer3 helicase blocks DNA heteroduplex extension in the 5' --> 3' direction. Our results support the idea that Mer3 helicase stabilizes nascent joint molecules via DNA heteroduplex extension to permit capture of the second processed end of a double-stranded DNA break, a step which is required for crossover recombinant product formation.     

Genetic Heterozygosity in Unreplicated Bacteriophage λ Recombinants

Bacteriophage crosses using density-labeled parents have been carried out under conditions restricting DNA synthesis. The parental material and genetic contributions to progeny manifesting recombination within a genetic interval sufficiently short to exhibit high negative interference have been examined. The unreplicated products of recombination isolated as phage particles appear to contain long continuous heteroduplex regions which are heterozygous for the closely linked markers. Recombination between closely linked markers seems to be the consequence of the removal of base-pair mismatches that are present within the heteroduplex regions. This localized reduction of heterozygosity within the heteroduplex regions that join the parental components of recombinant DNA molecules can account for high negative interference.     

Analysis of single-stranded DNA stability and damage-induced strand loss in mammalian cells using SV40-based shuttle vectors

The fate and stability of fully or partially single-stranded DNA molecules transfected into mammalian cells have been analysed. For this, we constructed a simian virus 40 (SV40)-based shuttle vector containing the f1 bacteriophage replication origin in the two possible orientations (pi SVF1-A and pi SVF1-B). This vector contains the SV40 origin of replication, the late viral genes and DNA sequences for replication and selection in Escherichia coli. It also carries the lacO sequence, which permits the analysis of plasmid stability. Single-stranded DNA from pi SVF1-A and pi SVF1-B were produced in bacteria and annealed in vitro to form a heteroduplex molecule. We showed that, in monkey kidney COS7 cells, single-stranded vectors replicate to form duplex molecules. After transfection of the three forms of molecules (single-stranded, heteroduplex or double-stranded), replicated DNA was rescued in E. coli. Vector stability was analysed by checking for plasmid rearrangements and screening for lacO mutants. The single-stranded pi SVF1 has a lower rearrangement level, while the spontaneous mutation frequency (on the lacO target) is in the same range as for the double-stranded vector. In contrast, the level of spontaneous mutagenesis is higher for the heteroduplex than for the single- and double-stranded forms. In addition, we found that replication of heteroduplex with one strand containing ultraviolet light-induced lesions yields progeny molecules in which the irradiated strand is mostly lost. This result indicates for the first time the specific loss of the damaged strand in mammalian cells.     

Effects of DNA Heterologies on Bacteriophage λ Recombination

Previous studies of bacteriophage λ recombination have provided indirect evidence that substantial sequence nonhomologies, such as insertions and deletions, may be included in regions of heteroduplex DNA. However, the direct products of heterology-containing heteroduplex DNA--heterozygous progeny phage--have not been observed. We have constructed a series of small insertion and deletion mutations in the cI gene to examine the possibility that small heterologies might be accommodated in heterozygous progeny phage. Genetic crosses were carried out between λcI(-) Oam29 and λcI(+) Pam80 under replication-restricted conditions. Recombinant O(+)P(+) progeny were selected on mutL hosts and tested for cI heterozygosity. Heterozygous recombinants were readily observed with crosses involving insertions of 4 to 19 base pairs (bp) in the cI gene. Thus, nonhomologies of at least 19 bp can be accommodated in regions of heteroduplex DNA during λ recombination. In contrast, when a cI insertion or deletion mutation of 26 bp was present, few of the selected recombinants were heterozygous for cI. Results using a substitution mutation, involving a 26-bp deletion with a 22-bp insertion, suggest that the low recovery of cI heterozygotes containing heterologies of 26 bp or more is due to a failure to encapsidate DNA containing heterologies of 26 bp or more into viable phage particles.     

The large loop repair and mismatch repair pathways of Saccharomyces cerevisiae act on distinct substrates during meiosis

During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplex DNA is formed when single-stranded DNAs from two homologs anneal as a consequence of strand invasion. If the two DNA strands differ in sequence, a mismatch will be generated. Mismatches in heteroduplex DNA are recognized and repaired efficiently by meiotic DNA mismatch repair systems. Components of two meiotic systems, mismatch repair (MMR) and large loop repair (LLR), have been identified previously, but the substrate range of these repair systems has never been defined. To determine the substrates for the MMR and LLR repair pathways, we constructed insertion mutations at HIS4 that form loops of varying sizes when complexed with wild-type HIS4 sequence during meiotic heteroduplex DNA formation. We compared the frequency of repair during meiosis in wild-type diploids and in diploids lacking components of either MMR or LLR. We find that the LLR pathway does not act on single-stranded DNA loops of <16 nucleotides in length. We also find that the MMR pathway can act on loops up to 17, but not >19, nucleotides in length, indicating that the two pathways overlap slightly in their substrate range during meiosis. Our data reveal differences in mitotic and meiotic MMR and LLR; these may be due to alterations in the functioning of each complex or result from subtle sequence context influences on repair of the various mismatches examined.     

Differential reaction kinetics, cleavage complex formation, and nonamer binding domain dependence dictate the structure-specific and sequence-specific nuclease activity of RAGs

During V(D)J recombination, RAG (recombination-activating gene) complex cleaves DNA based on sequence specificity. Besides its physiological function, RAG has been shown to act as a structure-specific nuclease. Recently, we showed that the presence of cytosine within the single-stranded region of heteroduplex DNA is important when RAGs cleave on DNA structures. In the present study, we report that heteroduplex DNA containing a bubble region can be cleaved efficiently when present along with a recombination signal sequence (RSS) in cis or trans configuration. The sequence of the bubble region influences RAG cleavage at RSS when present in cis. We also find that the kinetics of RAG cleavage differs between RSS and bubble, wherein RSS cleavage reaches maximum efficiency faster than bubble cleavage. In addition, unlike RSS, RAG cleavage at bubbles does not lead to cleavage complex formation. Finally, we show that the "nonamer binding region," which regulates RAG cleavage on RSS, is not important during RAG activity in non-B DNA structures. Therefore, in the current study, we identify the possible mechanism by which RAG cleavage is regulated when it acts as a structure-specific nuclease.     

Copy choice illegitimate DNA recombination

Precise excision of Tn10 and related transposons occurs by recombination between directly repeated 9 bp sequences that flank the transposon. The excision, which is a model for a class of illegitimate DNA recombination events, was stimulated 10(6) times by induction of single-stranded DNA synthesis, occurred during conversion of single-stranded DNA to double-stranded form, and entailed no transfer of physical material from parental to progeny molecules. We conclude that it occurred by copy choice DNA recombination and suggest that other illegitimate recombination events may occur by a similar mechanism.     

Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae

An enzyme catalyzing homologous pairing of DNA chains has been extensively purified from mitotic yeast. The most highly purified fractions are enriched for a polypeptide with a molecular mass of approximately 120 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein-dependent pairing of single-stranded DNAs requires a divalent cation (Mg2+ or Ca2+) but proceeds rapidly in the absence of any nucleoside triphosphates. The kinetics of reassociation are extremely rapid, with more than 60% of the single-stranded DNA becoming resistant to S1 nuclease within 1 min at a ratio of 1 protein monomer/50 nucleotides. The results of enzyme titration and DNA challenge experiments suggest that this protein does not act catalytically during renaturation but is required stoichiometrically. The protein promotes formation of joint molecules between linear M13 replicative form DNA (form III) containing short single-stranded tails and homologous single-stranded M13 viral DNA. Removal of approximately 50 nucleotides from the ends of the linear duplex using either exonuclease III (5' ends) or T7 gene 6 exonuclease (3' ends) activates the duplex for extensive strand exchange. Electron microscopic analysis of product molecules suggests that the homologous circular DNA initially associates with the single-stranded tails of the duplexes, and the heteroduplex region is extended with displacement of the noncomplementary strand. The ability of this protein to pair and to promote strand transfer using either exonuclease III or T7 gene 6 exonuclease-treated duplex substrates suggests that this activity promotes heteroduplex extension in a nonpolar fashion. The biochemical properties of the transferase are consistent with a role for this protein in heteroduplex joint formation during mitotic recombination in Saccharomyces cerevisiae.     

The DNA damage checkpoint allows recombination between divergent DNA sequences in budding yeast

In the early steps of homologous recombination, single-stranded DNA (ssDNA) from a broken chromosome invades homologous sequence located in a sister or homolog donor. In genomes that contain numerous repetitive DNA elements or gene paralogs, recombination can potentially occur between non-allelic/divergent (homeologous) sequences that share sequence identity. Such recombination events can lead to lethal chromosomal deletions or rearrangements. However, homeologous recombination events can be suppressed through rejection mechanisms that involve recognition of DNA mismatches in heteroduplex DNA by mismatch repair factors, followed by active unwinding of the heteroduplex DNA by helicases. Because factors required for heteroduplex rejection are hypothesized to be targets and/or effectors of the DNA damage response (DDR), a cell cycle control mechanism that ensures timely and efficient repair, we tested whether the DDR, and more specifically, the RAD9 gene, had a role in regulating rejection. We performed these studies using a DNA repair assay that measures repair by single-strand annealing (SSA) of a double-strand break (DSB) using homeologous DNA templates. We found that repair of homeologous DNA sequences, but not identical sequences, induced a RAD9-dependent cell cycle delay in the G2 stage of the cell cycle. Repair through a divergent DNA template occurred more frequently in RAD9 compared to rad9Δ strains. However, repair in rad9Δ mutants could be restored to wild-type levels if a G2 delay was induced by nocodazole. These results suggest that cell cycle arrest induced by the Rad9-dependent DDR allows repair between divergent DNA sequences despite the potential for creating deleterious genome rearrangements, and illustrates the importance of additional cellular mechanisms that act to suppress recombination between divergent DNA sequences.     

Primary products of break-induced recombination by Escherichia coli RecE pathway.

Alternative models for break-induced recombination predict different distributions of primary products. The double-stranded break-repair model predicts a noncrossover product and equimolar amounts of two crossover products. The one-end pairing model predicts two crossover products, but not necessarily in equimolar amounts, and the single-stranded annealing model predicts deletion of the fragment between the pairing sequences. Depending on the structure of the recombining substrate(s) and the nature of the resectioning step that precedes strand annealing, the single-stranded annealing mechanism would yield only one or both crossover products. We tested these predictions for the RecE recombination pathway of Escherichia coli. Nonreplicating intramolecular recombination substrates with a double-stranded break (DSB) within one copy of a direct repeat were released from chimera lambda phage by in vivo restriction, and the distribution of primary circular recombination products was determined. Noncrossover products were barely detectable, and the molar ratio of the two crossover products was proportional to the length ratio of the homologous ends flanking the DSB. These results suggest an independent pairing of each end with the intact homolog and argue against the double-stranded break-repair model. However, the results do not distinguish alternative pairing mechanisms (strand invasion and strand annealing). The kinetics of heteroduplex formation and heteroduplex strand polarity were investigated. Immediately following the DSB induction, heteroduplex formation was done by pairing the strands ending 3' at the break. A slow accumulation of the complementary heteroduplex made by the pairing of the strands ending 5' at the break (5' heteroduplexes) was observed at a larger stage. The observed bias in heteroduplex strand polarity depended on DSB induction at a specific site. The 5' heteroduplexes may have been generated by reciprocal strand exchange, pairing that is not strand specific, or strand-specific pairing induced at random breaks.     

Copy-choice illegitimate DNA recombination revisited.

Nearly precise excision of a transposon related to Tn10 from an Escherichia coli plasmid was used as a model to study illegitimate DNA recombination between short direct repeats. The excision was stimulated 100-1000 times by induction of plasmid single-stranded DNA synthesis and did not involve transfer of DNA from the parental to the progeny molecule. We conclude that it occurred by copy-choice DNA recombination, and propose that other events of recombination between short direct repeats might be a result of the same process.     

Double-strand cleavage at a two-base deletion mismatch in a DNA heteroduplex by nuclease S1

A two-base deletion mismatch was generated in a DNA heteroduplex by hybridization of two linear plasmid DNA molecules differing only by the presence of a two-base deletion in one of them. The heteroduplex was shown to be sensitive to double-strand cleavage by nuclease S1, thus demonstrating the potential value of single-stranded probes for the detection of polymorphisms in genomic DNA due to very small deletions.