Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity.     

Srs2 and Sgs1-Top3 suppress crossovers during double-strand break repair in yeast

Very few gene conversions in mitotic cells are associated with crossovers, suggesting that these events are regulated. This may be important for the maintenance of genetic stability. We have analyzed the relationship between homologous recombination and crossing-over in haploid budding yeast and identified factors involved in the regulation of crossover outcomes. Gene conversions unaccompanied by a crossover appear 30 min before conversions accompanied by exchange, indicating that there are two different repair mechanisms in mitotic cells. Crossovers are rare (5%), but deleting the BLM/WRN homolog, SGS1, or the SRS2 helicase increases crossovers 2- to 3-fold. Overexpressing SRS2 nearly eliminates crossovers, whereas overexpression of RAD51 in srs2Delta cells almost completely eliminates the noncrossover recombination pathway. We suggest Sgs1 and its associated topoisomerase Top3 remove double Holliday junction intermediates from a crossover-producing repair pathway, thereby reducing crossovers. Srs2 promotes the noncrossover synthesis-dependent strand-annealing (SDSA) pathway, apparently by regulating Rad51 binding during strand exchange.     

Genetic Evidence for Preferential Strand Transfer during Meiotic Recombination in Yeast

During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplexes are formed as an intermediate in the exchange process. In the formation of an asymmetric heteroduplex, one chromosome acts as a donor of a single DNA strand and the other acts as a recipient. We present genetic evidence that the nontranscribed strand is donated more frequently than the transcribed strand in spores that have an unrepaired mismatch at the HIS4 locus.     

Single-molecule views of MutS on mismatched DNA

Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides. The MSH mismatch-binding signal is then transmitted to the immediate downstream MutL homolog (MLH/PMS) MMR components and ultimately to a distant strand scission site where excision begins. The mechanism of signal transmission has been controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) and Polarization Total Internal Reflection Fluorescence (smP-TIRF) to examine the interactions and dynamic behaviors of single Thermus aquaticus MutS (TaqMutS) particles on mismatched DNA. We determined that TaqMutS forms an incipient clamp to search for a mismatch in ∼1 s intervals by 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while in continuous contact with the helical duplex DNA. When MutS encounters a mismatch it lingers for ∼3 s to exchange bound ADP for ATP (ADP → ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (∼10 min) that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The ATP-bound sliding clamps rotate freely while in discontinuous contact with the DNA. The visualization of a train of MSH proteins suggests that dissociation of ATP-bound sliding clamps from the mismatch permits multiple mismatch-dependent loading events. These direct observations have provided critical clues into understanding the molecular mechanism of MSH proteins during MMR.     

Uracil-DNA glycosylase affects mismatch repair efficiency in transformation and bisulfite-induced mutagenesis in Streptococcus pneumoniae.

The generalized mismatch repair system of Streptococcus pneumoniae (the Hex system) can eliminate base pair mismatches arising in heteroduplex DNA during transformation or by DNA polymerase errors during replication. Mismatch repair is most likely initiated at nicks or gaps. The present work was started to examine the hypothesis that strand discontinuities arising after removal of uracil by uracil DNA-glycosylase (Ung) can be utilised as strand discrimination signals. We show that mismatch repair efficiency is enhanced 3- to 6-fold when using uracil-containing DNA as donor in transformation. In order to assess the contribution of Ung to nascent strand discrimination for postreplication mismatch repair, we developed a positive selection procedure to isolate S. pneumoniae Ung- mutants. We succeeded in isolating Ung- mutants using this procedure based on chromosomal integration of uracil-containing hybrid DNA molecules. Cloning and characterization of the ung gene was achieved. Comparison of spontaneous mutation rates in strains either proficient or deficient in mismatch and/or uracil repair gave no support to the hypothesis that Ung plays a major role in targeting the Hex system to neosynthesized DNA strands. However Ung activity is responsible for the increased efficiency of mismatch repair observed in transformation with uracil-containing DNA. In addition Ung is involved in repair of bisulfite-treated transforming DNA.     

The gapped duplex DNA approach to oligonucleotide-directed mutation construction.

A simple and efficient method is described to introduce structurally pre-determined mutations into recombinant genomes of filamentous phage M13. The method rests on gapped duplex DNA (gdDNA) molecules of the phage M13 genome as the key intermediate. In this gdDNA, the (+) and the (shorter) (-) strand carry different genetic markers in such a way, that a rigorous selection can be applied for phage carrying the markers of the (-) strand. For introduction of the mutation, a synthetic oligonucleotide with partial homology to a target site within the single stranded DNA region is annealed to the gdDNA. The oligonucleotide subsequently becomes part of the (-) strand by enzymatic DNA gap filling and sealing. This physical linkage is preserved at the genetic level after transfection of a recipient E.coli strain deficient in DNA mismatch correction, so that the synthetic marker can be selected from the phage progeny independent from its potential phenotype. It is demonstrated that by this method mutants can be constructed with marker yields in excess of 70%.     

Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro

The ability of cell-free extracts to correct DNA mismatches has been demonstrated in both prokaryotes and eukaryotes. Such an assay requires a template containing both a mismatch and a strand discrimination signal, and the multi-step construction process can be technically difficult. We have developed a three-step procedure for preparing DNA heteroduplexes containing a site-specific nick. The mismatch composition, sequence context, distance to the strand signal, and the means for assessing repair in each strand are adjustable features built into a synthetic oligonucleotide. Controlled ligation events involving three of the four DNA strands incorporate the oligonucleotide into a circular template and generate the repair-directing nick. Mismatch correction in either strand of a prototype G·T mismatch was achieved by placing a nick 10–40 bp away from the targeted base. This proximity of nick and mismatch represents a setting where repair has not been well characterized, but the presence of a nick was shown to be essential, as was the MSH2/MSH6 heterodimer, although low levels of repair occurred in extract defective in each protein. All repair events were inhibited by a peptide that interacts with proliferating cell nuclear antigen and inhibits both mismatch repair and long-patch replication.     

Insertion and Deletion Mismatches Distant from the Target Position Improve Gene Correction with a Tailed Duplex

A 5′-tailed duplex (TD) DNA corrects a base-substitution mutation. In this study, the effects of insertion and deletion (indel) mismatches distant from the target position on the gene correction were examined. Three target plasmid DNAs with and without indel mismatches ～330 bases distant from the correction target position were prepared, and introduced into HeLa cells together with the TD. The indel mismatches improved the gene correction efficiency and specificity without sequence conversions at the indel mismatch site. These results suggested that the gene correction efficiency and specificity are increased when an appropriate second mismatch is introduced into the TD fragment.     

DNA template requirements for human mismatch repair in vitro

The human mismatch repair pathway is competent to correct DNA mismatches in a strand-specific manner. At present, only nicks are known to support strand discrimination, although the DNA end within the active site of replication is often proposed to serve this role. We therefore tested the competence of DNA ends or gaps to direct mismatch correction. Eight G.T templates were constructed which contained a nick or gap of 4, 28, or approximately 200 nucleotides situated approximately 330 bp away in either orientation. A competition was established in which the mismatch repair machinery had to compete with gap-filling replication and ligation activities for access to the strand discontinuity. Gaps of 4 or 28 nucleotides were the most effective strand discrimination signals for mismatch repair, whereas double strand breaks did not direct repair to either strand. To define the minimal spatial requirements for access to either the strand signal or mismatch site, the nicked templates were linearized close to either site and assayed. As few as 14 bp beyond the nick supported mismatch excision, although repair synthesis failed using 5'-nicked templates. Finally, asymmetric G.T templates with a remote nick and a nearby DNA end were repaired efficiently.     

Mismatch repair and the fidelity of genetic recombination

Two modes of mismatch repair are known to operate in bacteria: long-patch mismatch repair and very short patch mismatch repair. Very short patch mismatch repair systems act on a specific mismatch by conserving only one base pair. Therefore, when very short patch mismatch repair acts on heteroduplex recombination intermediates, it hyper-recombines specific markers by creating patchwork sequences, i.e., apparent multiple exchange events, on the repaired strand. Long-patch mismatch repair is antirecombinagenic, apparently by decomposing heteroduplex DNA or aborting its formation whenever well-recognized mismatches are formed by strand exchange between nonidentical parental sequences. It is postulated here that mismatch-stimulated antirecombination by long-patch mismatch repair is a "proofreading" system assuring high fidelity of homologous recombination. This accounts for chromosomal stability in eucaryotes (i.e., the rare occurrence of chromosomal aberrations and mitotic recombination versus the high frequency of precise sister chromatid exchange), suggests a role for diverged repetitive and other noncoding sequences as chromosomal antirecombination elements, and provides a molecular mechanism for speciation without the necessity of geographical separation.     

Human DNA mismatch repair in vitro operates independently of methylation status at CpG sites

Whereas in Escherichia coli DNA mismatch repair is directed to the newly synthesized strand due to its transient lack of adenine methylation, the molecular determinants of strand discrimination in eukaryotes are presently unknown. In mammalian cells, cytosine methylation within CpG sites may represent an analogous and mechanistically plausible means of targeting mismatch correction. Using HeLa nuclear extracts, we conducted a systematic analysis in vitro to determine whether cytosine methylation participates in human DNA mismatch repair. We prepared a set of A·C heteroduplex molecules that were either unmethylated, hemimethylated or fully methylated at CpG sequences and found that the methylation status persisted under the assay conditions. However, no effect on either the time course or the magnitude of mismatch repair events was evident; only strand discontinuities contributed to strand bias. By western analysis we demonstrated that the HeLa extract contained MED1 protein, which interacts with MLH1 and binds to CpG-methylated DNA; supplementation with purified MED1 protein was without effect. In summary, human DNA mismatch repair operates independently of CpG methylation status, and we found no evidence supporting a role for CpG hemimethylation as a strand discrimination signal.     

Correction of large mispaired DNA loops by extracts of Saccharomyces cerevisiae.

Single base mispairs and small loops are corrected by DNA mismatch repair, but little is known about the correction of large loops. In this paper, large loop repair was examined in nuclear extracts of yeast. Biochemical assays showed that repair activity occurred on loops of 16, 27, and 216 bases, whereas a G-T mispair and an 8-base loop were poorly corrected under these conditions. Two modes of loop repair were revealed by comparison of heteroduplexes that contained a site-specific nick or were covalently closed. A nick-stimulated repair mode directs correction to the discontinuous strand, regardless of which strand contains the loop. An alternative mode is nick-independent and preferentially removes the loop. Both outcomes of repair were largely eliminated when DNA replication was inhibited, suggesting a requirement for repair synthesis. Excision tracts of 100-200 nucleotides, spanning the position of the loop, were observed on each strand under conditions of limited DNA repair synthesis. Both repair modes were independent of the mismatch correction genes MSH2, MSH3, MLH1, and PMS1, as judged by activity in mutant extracts. Together the loop specificity and mutant results furnish evidence for a large loop repair pathway in yeast that is distinct from mismatch repair.     

The Effect of Nonhomologous DNA Sequences on Interplasmidic Recombination

The effect of nonhomologous DNA sequences at one or both sides of short genetic intervals on recombination within that interval was investigated, using an interplasmidic recombination system in Escherichia coli K-12. The recombining plasmids were derivatives of pBR322 and pACYC184, which share a 1330-nucleotide sequence that includes the tet gene. The genetic interval was defined by the HindIII and BamHI or BamHI and the SalI restriction endonuclease sites of this gene. The substantial differences between recombination frequencies measured within intervals bracketed or bounded on one side by major nonhomologies suggests that, in this system, strand exchange is polar and is blocked by major nonhomologies. This conclusion is substantiated by results of three-factor crosses and by structural analysis of recombination products. Results of two-factor crosses in recA genetic background and structural analysis of recombination products suggest that strand exchange occurs in the absence of a functional recA gene. "Opening" a bracketed BamHI-SalI genetic interval of the tet gene, at the SalI site, by substituting a major insertion with a short deletion, results in an increase in recombination frequency, within this genetic interval, which is greater than expected on the basis of the ratio of the length of homology on the two sides of the SalI site. This observation suggests that a genetic element that may affect rate of recombination initiation, polarity of strand exchange, template specificity in mismatch repair or more than one of these events may be present on the outer side of the SalI site of the tet gene.     

Antipairing and strand transferase activities of E. coli helicase II (UvrD).

The product of the uvrD gene of Escherichia coli, UvrD (helicase II), is known to be involved in methyl-directed mismatch repair, transposon excision and uvrABC excision repair. In conjugational crosses, various uvrD mutants have been reported to result in higher, lower or unaffected recombination frequencies. In an attempt to clarify the role of UvrD in recombination, we have studied in vitro its effects on two key reactions driven by RecA, homologous pairing and strand exchange. We show here that UvrD efficiently prevents or reverses RecA-mediated homologous pairing. Unexpectedly, we also found that it can stimulate RecA-driven branch migration and even catalyze strand exchange in the absence of RecA. A possible in vivo role for these antagonistic activities is discussed.     

Problems and Paradigms: Relating biochemistry to biology: How the recombinational repair function of RecA protein is manifested in its molecular properties

The multiple activities of the RecA protein in DNA metabolism have inspired over a decade of research in dozens of laboratories around the world. This effort has nevertheless failed to yield an understanding of the mechanism of several RecA protein-mediated processes, the DNA strand exchange reactions prominent among them. The major factors impeding progress are the invalid constraints placed upon the problem by attempting to understand RecA protein-mediated DNA strand exchange within the context of an inappropriate biological paradigm – namely, homologous genetic recombination as a mechanism for generating genetic diversity. In this essay I summarize genetic and biochemical data demonstrating that RecA protein evolved as the central component of a recombinational DNA repair system, with the generation of genetic diversity being a sometimes useful byproduct, and review the major in vitro activities of RecA protein from a repair perspective. While models proposed for both recombination and recombinational repair often make use of DNA strand cleavage and transfer steps that appear to be quite similar, the molecular and thermodynamic requirements of the two processes are very different. The recombinational repair function provides a much more logical and informative framework for thinking about the biochemical properties of RecA and the strand exchange reactions it facilitates.     

Suppression of homologous and homeologous recombination by the bacterial MutS2 protein

In addition to their role in DNA repair, recombination events are associated with processes aimed at providing the genetic variability needed for adaptation and evolution of a population. In bacteria, recombination is involved in the appearance of new variants by allowing the incorporation of exogenous DNA or the reshuffling of endogenous sequences. Here we show that HpMutS2, a protein belonging to the MutS2 family in Helicobacter pylori, is not involved in mismatch repair but inhibits homologous and homeologous recombination. Disruption of HpmutS2 leads to an increased efficiency of exogenous DNA incorporation. HpMutS2 has a selective affinity for DNA structures mimicking recombination intermediates with no specificity for homoduplex DNA or mismatches. The purified protein has an ATPase activity stimulated by the same DNA structures. Finally, we show that HpMutS2 inhibits DNA strand exchange reactions in vitro. Thus, MutS2 proteins are candidates for controlling recombination and therefore genetic diversity in bacteria.     

Redundant exonuclease involvement in Escherichia coli methyl-directed mismatch repair.

Previous biochemical analysis of Escherichia coli methyl-directed mismatch repair implicates three redundant single-strand DNA-specific exonucleases (RecJ, ExoI, and ExoVII) and at least one additional unknown exonuclease in the excision reaction (Cooper, D. L., Lahue, R. S., and Modrich, P. (1993) J. Biol. Chem. 268, 11823-11829). We show here that ExoX also participates in methyl-directed mismatch repair. Analysis of the reaction with crude extracts and purified components demonstrated that ExoX can mediate repair directed from a strand signal 3' of a mismatch. Whereas extracts of all possible single, double, and triple exonuclease mutants displayed significant residual mismatch repair, extracts deficient in RecJ, ExoI, ExoVII, and ExoX exonucleases were devoid of normal repair activity. The RecJ(-) ExoVII(-) ExoI(-) ExoX(-) strain displayed a 7-fold increase in mutation rate, a significant increase, but less than that observed for other blocks of the mismatch repair pathway. This elevation is epistatic to deficiency for MutS, suggesting an effect via the mismatch repair pathway. Our other work (Burdett, V., Baitinger, C., Viswanathan, M., Lovett, S. T., and Modrich, P. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 6765-6770) suggests that mutants are under-recovered in the exonuclease-deficient strain due to loss of viability that is triggered by mismatched base pairs in this genetic background. The availability of any one exonuclease is enough to support full mismatch correction, as evident from the normal mutation rates of all triple mutants. Because three of these exonucleases possess a strict polarity of digestion, this suggests that mismatch repair can occur exclusively from a 3' or a 5' direction to the mismatch, if necessary.     

Mismatch Repair-Induced Meiotic Recombination Requires the Pms1 Gene Product

The presence of multiple heterologies in a 9-kilobase (kb) interval results in a decrease in meiotic crossovers from 26.0% to 10.1%. There is also an increase from 3.5% to 11.1% in gene conversions and ectopic recombinations between the flanking homologous MAT loci. The hypothesis that mismatch repair of heteroduplex DNA containing several heterologies would lead to a second round of recombination has now been tested by examining the effect of a mutation that reduces mismatch correction. The repair-defective pms1-1 allele restores the pattern of recombination to nearly that seen in congenic diploids without the heterologies. Mismatch repair-induced recombination causes a significant increase in MAT conversions and ectopic recombination events with as few as two heterozygosities separated by 0.3-0.7 kb, but not when the mismatches are separated by greater than 1 kb. The frequency of these events depends on both the number and position of the heterozygosities relative to the flanking homologous MAT loci used to detect the events. The creation of recombinogenic lesions by mismatch repair in yeast could be analogous to the creation of recombinogenic lesions in dam- Escherichia coli. We suggest that the repair of heteroduplex DNA containing multiple mismatches may produce chromosomal rearrangements and gamete inviability when naturally polymorphic chromosomes undergo meiotic recombination.     

A molecular model for RecA-promoted strand exchange via parallel triple-stranded helices.

A number of studies have concluded that strand exchange between a RecA-complexed DNA single strand and a homologous DNA duplex occurs via a single-strand invasion of the minor groove of the duplex. Using molecular modeling, we have previously demonstrated the possibility of forming a parallel triple helix in which the single strand interacts with the intact duplex in the minor groove, via novel base interactions (Bertucat et al., J. Biomol. Struct. Dynam. 16:535-546). This triplex is stabilized by the stretching and unwinding imposed by RecA. In the present study, we show that the bases within this triplex are appropriately placed to undergo strand exchange. Strand exchange is found to be exothermic and to result in a triple helix in which the new single strand occupies the major groove. This structure, which can be equated to so-called R-form DNA, can be further stabilized by compression and rewinding. We are consequently able to propose a detailed, atomic-scale model of RecA-promoted strand exchange. This model, which is supported by a variety of experimental data, suggests that the role of RecA is principally to prepare the single strand for its future interactions, to guide a minor groove attack on duplex DNA, and to stabilize the resulting, stretched triplex, which intrinsically favors strand exchange. We also discuss how this mechanism can incorporate homologous recognition.     

Effects of Holliday junction position on Xer-mediated recombination in vitro.

Site-specific recombination mediated by XerC and XerD functions in the segregation of circular replicons in Escherichia coli. A key feature of most models of recombination for the family of recombinases to which XerC and XerD belong is that a Holliday junction forms at the position of the first pair of recombinase-mediated strand exchanges and then branch migrates 6-8 bp to the position of the second pair of strand exchanges. We have tested this hypothesis for Xer recombination by studying the effects of junction position on XerC-mediated strand exchange in vitro. Recombination of synthetic Holliday junction substrates in which junction mobility was constrained to a region extending over or removed away from the normal cleavage and exchange point was analysed. All substrates undergo strand cleavage at the normal position. We infer that the Holliday junction need not be at this position during strand cleavage and exchange. With substrates in which the Holliday junction is constrained to a region away from the XerC-mediated cleavage point, strand exchange generates products with the predicted mispaired bases.