Characterization of recombination intermediates from DNA injected into Xenopus laevis oocytes: evidence for a nonconservative mechanism of homologous recombination.

Homologous recombination between DNA molecules injected into Xenopus laevis oocyte nuclei is extremely efficient if injected molecules have overlapping homologous ends. Earlier work demonstrated that ends of linear molecules are degraded by a 5'----3' exonuclease activity, yielding 3' tails that participate in recombination. Here, we have characterized intermediates further advanced along the recombination pathway. The intermediates were identified by their unique electrophoretic and kinetic properties. Two-dimensional gel electrophoresis and hybridization with oligonucleotide probes showed that the intermediates had heteroduplex junctions within their homologous overlaps in which strands ending 3' were full length and those ending 5' were shortened. Additional characterization suggested that these intermediates had formed by the annealing of complementary 3' tails. Annealed junctions made in vitro were rapidly processed to products, indicating that they are on the normal recombination pathway. These results support a nonconservative, single-strand annealing mode of recombination. This recombination mechanism appears to be shared by many organisms, including bacteria, fungi, plants, and mammals.     

Mechanisms of overlap formation in nonhomologous DNA end joining.

Rejoining of nonhomologous DNA termini plays a central role in processes of illegitimate recombination. In Xenopus egg extracts, DNA ends with noncomplementary 4-nucleotide antiparallel single-strand protrusions are assumed to be joined by formation of short mismatched overlap intermediates. The extents of these overlaps may be set by single fortuitously matching base pairs and determine the patterns of subsequent gap filling and nick ligation. Under conditions of alternative overlap settings, rules for the most probable joining pathway and the effects of mismatches on junction formation were analyzed. We show that in certain cases, fill-in and ligation converting overlap intermediates into covalently closed junctions may proceed in the presence of unrepaired mismatches, whereas in other cases, completion of junction formation is preceded by removal of mismatches. Results are discussed in relation with "alignment" proteins postulated to structurally support overlap heteroduplexes during junction formation.     

Effect of terminal nonhomologies on homologous recombination in Xenopus laevis oocytes.

Homologous recombination of linear DNA molecules in Xenopus laevis oocytes is very efficient. The predictions of molecular models for this recombination process were tested with substrates with terminal nonhomologies (nonhomologous sequences). It was found that nonhomologies on one or both ends of an otherwise efficient substrate substantially reduced the yield of recombination products. In the case of a single nonhomology, inhibition was observed for all lengths of nonhomology, from 60 to 1,690 bp, being most dramatic for the longer blocks. Examination of time courses of recombination showed that the blocks were largely kinetic; that is, substrates with short nonhomologies eventually yielded substantial levels of completed products. Intermediates that accumulated after the injection of end-blocked substrates were characterized by two-dimensional gel electrophoresis and hybridization with strand-specific oligonucleotide probes. These blocked intermediates were shown to have base-paired junctions, but resolution was prevented by the failure to remove the 3'-ending strand of the original nonhomology. Continuing exonuclease action created a single-strand gap adjacent to the position of the persistent nonhomology. In contrast, the strand that included the unblocked side of the junction could be sealed. These results are consistent with a nonconservative, resection-annealing mechanism of homologous recombination in the oocytes and suggest the absence of any activity that can efficiently remove 3' tails.     

DNA double strand break repair and crossing over mediated by RuvABC resolvase and RecG translocase

DNA double-strand breaks threaten the stability of the genome, and yet are induced deliberately during meiosis in order to provoke homologous recombination and generate the crossovers needed to promote faithful chromosome transmission. Crossovers are secured via biased resolution of the double Holliday junction intermediates formed when both ends of the broken chromosome engage an intact homologue. To investigate whether the enzymes catalysing branch migration and resolution of Holliday junctions are directed to favour production of either crossover or noncrossover products, we engineered a genetic system based on DNA breakage induced by the I-SceI endonuclease to detect analogous exchanges in Escherichia coli where the enzymology of recombination is more fully understood. Analysis of the recombinants generated revealed approximately equal numbers of crossover and noncrossover products, regardless of whether repair is mediated via RecG, RuvABC, or the RusA resolvase. We conclude that there little or no control of crossing over at the level of Holliday junction resolution. Thus, if similar resolvases function during meiosis, additional factors must act to bias recombination in favour of crossover progeny.     

Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells.

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.     

Mechanisms of nonhomologous recombination in mammalian cells.

The primary mechanism of nonhomologous recombination in transfected DNA involves breakage followed by end joining. To probe the joining step in more detail, linear simian virus 40 genomes with mismatched ends were transfected into cultured monkey cells, and individual viable recombinants were analyzed. The transfected genomes carried mismatched ends as a result of cleavage with two restriction enzymes, the recognition sites of which are located in the intron of the gene encoding the T antigen. Because the T antigen gene was split by this cleavage, the transfected genomes were inert until activated by cell-mediated end joining. Clonal descendants of the original recombinants were isolated from 122 plaques and were grouped into four classes based on the electrophoretic mobility of the junction fragment. The structures of representative junctions were determined by nucleotide sequencing. The spectrum of nonhomologous junctions analyzed here along with a large number of previously reported junctions suggest that there are two mechanisms for the linkage of DNA molecules: (i) direct ligation of ends and (ii) repair synthesis primed by terminal homologies of a few nucleotides. A paired-priming model of nonhomologous recombination is discussed.     

Multiple pathways for repair of DNA double-strand breaks in mammalian chromosomes

To study repair of DNA double-strand breaks (DSBs) in mammalian chromosomes, we designed DNA substrates containing a thymidine kinase (TK) gene disrupted by the 18-bp recognition site for yeast endonuclease I-SceI. Some substrates also contained a second defective TK gene sequence to serve as a genetic donor in recombinational repair. A genomic DSB was induced by introducing endonuclease I-SceI into cells containing a stably integrated DNA substrate. DSB repair was monitored by selection for TK-positive segregants. We observed that intrachromosomal DSB repair is accomplished with nearly equal efficiencies in either the presence or absence of a homologous donor sequence. DSB repair is achieved by nonhomologous end-joining or homologous recombination, but rarely by nonconservative single-strand annealing. Repair of a chromosomal DSB by homologous recombination occurs mainly by gene conversion and appears to require a donor sequence greater than a few hundred base pairs in length. Nonhomologous end-joining events typically involve loss of very few nucleotides, and some events are associated with gene amplification at the repaired locus. Additional studies revealed that precise religation of DNA ends with no other concomitant sequence alteration is a viable mode for repair of DSBs in a mammalian genome.     

Control of crossing over

The Holliday junction is a central intermediate in homologous recombination. It consists of a four-way structure that can be resolved by cleavage to give either the crossover or noncrossover products observed. We show here that the formation of these products is controlled by the E. coli resolvasome (RuvABC) in such way that double-strand break repair (DSBR) leads to crossing over and single-strand gap repair (SSGR) does not lead to crossing over. We argue that the positioning of the RuvABC complex and its consequent direction of junction-cleavage is not random. In fact, the action of the RuvABC complex avoids crossing over in the most commonly predicted situations where Holliday junctions are encountered in DNA replication and repair. Our observations suggest that the positioning of the resolvasome may provide a general biochemical mechanism by which cells can control crossing over in recombination.     

ruvA and ruvB mutants specifically impaired for replication fork reversal

Replication fork reversal (RFR) is a reaction that takes place in Escherichia coli at replication forks arrested by the inactivation of a replication protein. Fork reversal involves the annealing of the leading and lagging strand ends; it results in the formation of a Holliday junction adjacent to DNA double-strand end, both of which are processed by recombination enzymes. In several replication mutants, replication fork reversal is catalysed by the RuvAB complex, originally characterized for its role in the last steps of homologous recombination, branch migration and resolution of Holliday junctions. We present here the isolation and characterization of ruvA and ruvB single mutants that are impaired for RFR at forks arrested by the inactivation of polymerase III, while they remain capable of homologous recombination. The positions of the mutations in the proteins and the genetic properties of the mutants suggest that the mutations affect DNA binding, RuvA-RuvB interaction and/or RuvB-helicase activity. These results show that a partial RuvA or RuvB defect affects primarily RFR, implying that RFR is a more demanding reaction than Holliday junction resolution.     

Direct repeats of the herpes simplex virus a sequence promote nonconservative homologous recombination that is not dependent on XPF/ERCC4.

We have examined mechanisms of recombination in mammalian cells infected with herpes simplex virus type 1 (HSV-1). Amplification of plasmids containing a viral origin of replication, oriS, in cells superinfected with HSV-1 revealed that linear DNA could be efficiently converted to templates for replication. Two distinct pathways were observed: imprecise end joining and nonconservative homologous recombination. We noted that direct repeats of the viral a sequence promoted efficient nonconservative homologous recombination in BHK cells as well as human repair-proficient 1BR.3N cells and xeroderma pigmentosum group F (XP-F) cells. The reaction gave rise to functional a sequences supporting the formation of defective viruses. It did not seem to proceed by single-strand annealing since it occurred in the absence of XPF/ERCC4, the mammalian homolog of the Rad1 endonuclease from Saccharomyces cerevisiae. In contrast, direct repeats of a 161-bp nonviral sequence did not take part in nonconservative homologous recombination in XP-F cells. Our results suggest that homologous recombination may be involved in the circularization of viral genomes. Furthermore, they demonstrate that amplification of recombination products supported by HSV-1 allows a direct examination of pathways for double-strand-break repair in human cells.     

The structure-specific endonuclease Ercc1-Xpf is required for targeted gene replacement in embryonic stem cells.

The Ercc1-Xpf heterodimer, a highly conserved structure-specific endonuclease, functions in multiple DNA repair pathways that are pivotal for maintaining genome stability, including nucleotide excision repair, interstrand crosslink repair and homologous recombination. Ercc1-Xpf incises double-stranded DNA at double-strand/single-strand junctions, making it an ideal enzyme for processing DNA structures that contain partially unwound strands. Here we demonstrate that although Ercc1 is dispensable for recombination between sister chromatids, it is essential for targeted gene replacement in mouse embryonic stem cells. Surprisingly, the role of Ercc1-Xpf in gene targeting is distinct from its previously identified role in removing nonhomologous termini from recombination intermediates because it was required irrespective of whether the ends of the DNA targeting constructs were heterologous or homologous to the genomic locus. Our observations have implications for the mechanism of gene targeting in mammalian cells and define a new role for Ercc1-Xpf in mammalian homologous recombination. We propose a model for the mechanism of targeted gene replacement that invokes a role for Ercc1-Xpf in making the recipient genomic locus receptive for gene replacement.     

Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes

Homologous recombination is required for maintaining genomic integrity by functioning in high-fidelity repair of DNA double-strand breaks and other complex lesions, replication fork support, and meiotic chromosome segregation. Joint DNA molecules are key intermediates in recombination and their differential processing determines whether the genetic outcome is a crossover or non-crossover event. The Holliday model of recombination highlights the resolution of four-way DNA joint molecules, termed Holliday junctions, and the bacterial Holliday junction resolvase RuvC set the paradigm for the mechanism of crossover formation. In eukaryotes, much effort has been invested in identifying the eukaryotic equivalent of bacterial RuvC, leading to the discovery of a number of DNA endonucleases, including Mus81?CMms4/EME1, Slx1?CSlx4/BTBD12/MUS312, XPF?CERCC1, and Yen1/GEN1. These nucleases exert different selectivity for various DNA joint molecules, including Holliday junctions. Their mutant phenotypes and distinct species-specific characteristics expose a surprisingly complex system of joint molecule processing. In an attempt to reconcile the biochemical and genetic data, we propose that nicked junctions constitute important in vivo recombination intermediates whose processing determines the efficiency and outcome (crossover/non-crossover) of homologous recombination.     

Evidence for biased holliday junction cleavage and mismatch repair directed by junction cuts during double-strand-break repair in mammalian cells

In mammalian cells, several features of the way homologous recombination occurs between transferred and chromosomal DNA are consistent with the double-strand-break repair (DSBR) model of recombination. In this study, we examined the segregation patterns of small palindrome markers, which frequently escape mismatch repair when encompassed within heteroduplex DNA formed in vivo during mammalian homologous recombination, to test predictions of the DSBR model, in particular as they relate to the mechanism of crossover resolution. According to the canonical DSBR model, crossover between the vector and chromosome results from cleavage of the joint molecule in two alternate sense modes. The two crossover modes lead to different predicted marker configurations in the recombinants, and assuming no bias in the mode of Holliday junction cleavage, the two types of recombinants are expected in equal frequency. However, we propose a revision to the canonical model, as our results suggest that the mode of crossover resolution is biased in favor of cutting the DNA strands upon which DNA synthesis is occurring during formation of the joint molecule. The bias in junction resolution permitted us to examine the potential consequences of mismatch repair acting on the DNA breaks generated by junction cutting. The combination of biased junction resolution with both early and late rounds of mismatch repair can explain the marker patterns in the recombinants.     

The phage T4 protein UvsW drives Holliday junction branch migration

The phage T4 UvsW protein has been shown to play a crucial role in the switch from origin-dependent to recombination-dependent replication in T4 infections through the unwinding of origin R-loop initiation intermediates. UvsW also functions with UvsX and UvsY to repair damaged DNA through homologous recombination, and, based on genetic evidence, has been proposed to act as a Holliday junction branch migration enzyme. Here we report the purification and characterization of UvsW. Using oligonucleotide-based substrates, we confirm that UvsW unwinds branched DNA substrates, including X and Y structures, but shows little activity in unwinding linear duplex substrates with blunt or single-strand ends. Using a novel Holliday junction-containing substrate, we also demonstrate that UvsW promotes the branch migration of Holliday junctions efficiently through more than 1000 bp of DNA. The ATP hydrolysis-deficient mutant protein, UvsW-K141R, is unable to promote Holliday junction branch migration. However, both UvsW and UvsW-K141R are capable of stabilizing Holliday junctions against spontaneous branch migration when ATP is not present. Using two-dimensional agarose gel electrophoresis we also show that UvsW acts on T4-generated replication intermediates, including Holliday junction-containing X-shaped intermediates and replication fork-shaped intermediates. Taken together, these results strongly support a role for UvsW in the branch migration of Holliday junctions that form during T4 recombination, replication, and repair.     

No braiding of Holliday junctions in positively supercoiled DNA molecules

The Holliday junction is a prominent intermediate in genetic recombination that consists of four double helical arms of DNA flanking a branch point. Under many conditions, the Holliday junction arranges its arms into two stacked domains that can be oriented so that genetic markers are parallel or antiparallel. In this arrangement, two strands retain a helical conformation, and the other two strands effect the crossover between helical domains. The products of recombination are altered by a crossover isomerization event, which switches the strands fulfilling these two roles. It appears that effecting this switch from the parallel conformation by the simplest mechanism results in braiding the crossover strands at the branch point. In previous work we showed by topological means that a short, parallel, DNA double crossover molecule with closed ends did not braid its branch point; however, that molecule was too short to adopt the necessary positively supercoiled topology. Here, we have addressed the same problem using a larger molecule of the same type. We have constructed a parallel DNA double crossover molecule with closed ends, containing 14 double helical turns in each helix between its crossover points. We have prepared this molecule in a relaxed form by simple ligation and in a positively supercoiled form by ligation in the presence of netropsin. The positively supercoiled molecule is of the right topology to accommodate braiding. We have compared the relaxed and supercoiled versions for their responses to probes that include hydroxyl radicals, KMnO4, the junction resolvases endonuclease VII and RuvC, and RuvC activation of KMNO4 sensitivity. In no case did we find evidence for a braid at the crossover point. We conclude that Holliday junctions do not braid at their branch points, and that the topological problem created by crossover isomerization in the parallel conformation is likely to be solved by distributing the stress over the helices that flank the branch point.     

An archaeal Holliday junction resolving enzyme from Sulfolobus solfataricus exhibits unique properties

The rearrangement and repair of DNA by homologous recombination often involves the creation of Holliday junctions, which must be cleaved by junction-specific endonucleases to yield recombinant duplex DNA products. Holliday junction resolving enzymes are a ubiquitous class of proteins with diverse structural and mechanistic characteristics. We have characterised an endonuclease (Hje) from the thermophilic crenarchaeote Sulfolobus solfataricus that exhibits a high degree of specificity for Holliday junctions via an apparently novel mechanism. Hje resolves four-way DNA junctions by the introduction of paired nicks in a reaction that is independent of the local nucleotide sequence, but is restricted solely to strands that are continuous in the stacked-X form of the junction. Three-way DNA junctions are cleaved only when the presence of a bulge in one strand allows the junction to stack in an analogous manner to four-way junctions. These properties differentiate Hje from all other known junction resolving enzymes.     

Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination

The characterization of plasmid-genomic DNA junctions following plant transformation has established links between DNA double-strand break repair (DSBR), illegitimate recombination and plasmid DNA integration. The limited information on plasmid-plasmid junctions in plants comes from the dicot species tobacco and Arabidopsis. We analyzed 12 representative transgenic rice lines, carrying a range of transforming plasmid rearrangements, which predominantly reflected microhomology mediated illegitimate recombination involving short complementary patches at the recombining ends. Direct end-ligation, in the absence of homology between the recombining molecules, occurred only rarely. Filler DNA was found at some of the junctions. Short, purine-rich tracts were present, either at the junction site or in the immediate flanking regions. Putative DNA topoisomerase I binding sites were clustered around the junctions. Although different regions of the transforming plasmid were involved in plasmid-plasmid recombination, we showed that a 19 bp palindromic sequence, including the TATA box of the CaMV 35S promoter, acted as a recombination hotspot. The purine-rich half of the palindromic sequence was specifically involved at the recombination junctions. This recombination hotspot is located within the 'highly recombinogenic' region of the full-length CaMV RNA that has been shown to promote viral recombination in dicot plants. Clustering of plasmid recombination events in this highly recombinogenic region, even in the absence of viral enzymes and other cis-acting elements proves that the plant cellular machinery alone is sufficient to recognize and act on these viral sequences. Our data also show the similarity between mechanisms underlying junction formation in dicot and monocot plants transformed using different procedures.     

Enzymatic formation of biparental figure-eight molecules from plasmid DNA and their resolution in E. coli

A key intermediate in general genetic recombination is a structure in which two double-stranded DNA molecules are covalently linked by a single-strand crossover characteristic of a Holliday junction. When the DNA molecules are circular, the recombinant structures take the form of a figure eight. We have used purified E. coli enzymes to construct biparental figure-eight DNA molecules in vitro from the DNA of two partially homologous plasmids. When purified figure-eight structures are transfected into recA- E. coli cells, they are resolved to produce monomeric or dimeric plasmid progeny, apparently by the cutting and joining of the Holliday crossover. The maturation of figure-eight molecules in bacteria is characterized by the formation and recovery of both parental and recombinant types, cross-over at a frequency of up to 50% and the capability for mismatch repair at regions of hybrid DNA. In these three regards, the products of figure-eight maturation resemble recombinant chromosomes formed at meiosis. These observations show that biparental figure eights behave as recombination intermediates that can be resolved into mature recombinants without need for a functional recA+ gene product.     

Mechanism and control of recombination in fungi.

In fungi, most mitotic recombination and at least some meiotic recombination appear to stem from a process of double-strand break repair. During this repair, recombination occurs by conversion caused by the process of double-strand gap filling, by conversion related to heteroduplex formation where homologous molecules interact by complementary base pairing, and by crossing-over which is probably an occasional byproduct of the repair process. From a review of the genetic and biochemical data and the published models of the process of recombination, the following view emerges: broken ends may be acted upon by nucleases and helicases to produce a recombinagenic end which may have both 3' and 5' single-stranded tails. These postulated split-ends may then act independently to find regions of homology with which to react. Invasion by both ends forms two splice-junctions which prime DNA synthesis towards each other to replace lost information, using the homologous sequences as templates. This process would lead to a structure which consists of a double Holliday junction which may be resolved endonucleolytically, sometimes giving a crossover, or by another means such as the action of topoisomerase, to dissolve the structure without a crossover having been formed.     

T-DNA recombination and replication in maize cells

T-DNA recombination and replication was analyzed in 'black mexican sweet' (BMS) cells transformed with T-DNAs containing the replication system from wheat dwarf virus (WDV). Upon recombination between the T-DNA ends, a promoterless marker gene (gusA) was activated. Activation of the recombination marker gene was delayed and increased exponentially over time, suggesting that recombination and amplification of the T-DNA occurred in maize cells. Mutant versions of the viral initiator gene (rep), known to be defective in the replication function, failed to generate recoverable recombinant T-DNA molecules. Circularization of T-DNA by the FLP/FRT site-specific recombination system and/or homologous recombination was not necessary to recover circular T-DNAs. However, replicating T-DNAs appeared to be suitable substrates for site-specific and homologous recombination. Among 33 T-DNA border junctions sequenced, only one pair of identical junction sites was found implying that the population of circular T-DNAs was highly heterogenous. Since no circular T-DNA molecules were detected in treatments without rep, it suggested that T-DNA recombination was linked to replication and might have been stimulated by this process. The border junctions observed in recombinant T-DNA molecules were indicative of illegitimate recombination and were similar to left-border recombination of T-DNA into the genome after Agro-mediated plant transformation. However, recombination between T-DNA molecules differed from T-DNA/genomic DNA junction sites in that few intact right borders were observed. The replicating T-DNA molecules did not enhance genomic random integration of T-DNA in the experimental configuration used for this study.