Mismatch Repair by Efficient Nick-Directed, and Less Efficient Mismatch-Specific, Mechanisms in Homologous Recombination Intermediates in Chinese Hamster Ovary Cells

Repair of single-base mismatches formed in recombination intermediates in vivo was investigated in Chinese hamster ovary cells. Extrachromosomal recombination was stimulated by double-strand breaks (DSBs) introduced into regions of shared homology in pairs of plasmid substrates heteroallelic at 11 phenotypically silent mutations. Recombination was expected to occur primarily by single-strand annealing, yielding predicted heteroduplex DNA (hDNA) regions with three to nine mismatches. Product spectra were consistent with hDNA only occurring between DSBs. Nicks were predicted on opposite strands flanking hDNA at positions corresponding to original DSB sites. Most products had continuous marker patterns, and observed conversion gradients closely matched predicted gradients for repair initiated at nicks, consistent with an efficient nick-directed, excision-based mismatch repair system. Discontinuous patterns, seen in ~10% of products, and deviations from predicted gradients provided evidence for less efficient mismatch-specific repair, including G-A -> G-C specific repair that may reflect processing by a homologue of Escherichia coli MutY. Mismatch repair was >80% efficient, which is higher than seen previously with covalently closed, artificial hDNA substrates. Products were found in which all mismatches were repaired in a single tract initiated from one or the other nick. We also observed products resulting from two tracts of intermediate length initiated from two nicks.     

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.     

Measurements of excision repair tracts formed during meiotic recombination in Saccharomyces cerevisiae.

During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplexes are formed at a high frequency between HIS4 genes located on homologous chromosomes. Using mutant alleles of the HIS4 gene that result in poorly repaired mismatches in heteroduplex DNA, we find that heteroduplexes often span a distance of 1.8 kb. In addition, we show that about one-third of the repair tracts initiated at well-repaired mismatches extend 900 bp.     

Patterns of heteroduplex formation associated with the initiation of meiotic recombination in the yeast Saccharomyces cerevisiae

The double-strand break repair (DSBR) model of recombination predicts that heteroduplexes will be formed in regions that flank the double-strand break (DSB) site and that the resulting intermediate is resolved to generate either crossovers or noncrossovers for flanking markers. Previous studies in Saccharomyces cerevisiae, however, failed to detect heteroduplexes on both sides of the DSB site. Recent physical studies suggest that some recombination events involve heterodupex formation by a mechanism, synthesis-dependent strand annealing (SDSA), that is inherently asymmetric with respect to the DSB site and that leads exclusively to noncrossovers of flanking markers. Below, we demonstrate that many of the recombination events initiated at the HIS4 recombination hotspot are consistent with a variant of the DSBR model in which the extent of heteroduplex on one side of the DSB site is much greater than that on the other. Events that include only one flanking marker in the heteroduplex (unidirectional events) are usually resolved as noncrossovers, whereas events that include both flanking markers (bidirectional events) are usually resolved as crossovers. The unidirectional events may represent SDSA, consistent with the conclusions of others, although other possibilities are not excluded. We also show that the level of recombination reflects the integration of events initiated at several different DSB sites, and we identify a subset of gene conversion events that may involve break-induced replication (BIR) or repair of a double-stranded DNA gap.     

Heteroduplex repair in extracts of human HeLa cells

A general repair process for DNA heteroduplexes has been detected in HeLa cell extracts. Using a variety of M13mp2 DNA substrates containing single-base mismatches and extra nucleotides, extensive repair is observed after incubation with HeLa cell cytoplasmic extracts and subsequent transfection of bacterial cells with the treated DNA. Most, but not all, mispairs as well as two frameshift heteroduplexes are repaired efficiently. Parallel measurements of repair in HeLa extracts and in Escherichia coli suggest that repair specificities are similar for the two systems. The presence of a nick in the molecule is required for efficient repair in HeLa cell extracts, and the strand containing the nick is the predominantly repaired strand. Mismatch-dependent DNA synthesis is observed when radiolabeled restriction fragments, produced by reaction of the extract with heteroduplex and homoduplex molecules, are compared. Specific labeling of fragments, representing a region of approximately 1,000 base pairs and containing the nick and the mismatch, is detected for the heteroduplex substrate but not the homoduplex. The repair reaction is complete after 20 min and requires added Mg2+, ATP, and an ATP-regenerating system, but not dNTPs, which are present at sufficient levels in the extract. An inhibitor of DNA polymerase beta, dideoxythimidine 5'-triphosphate, does not inhibit mismatch-specific DNA synthesis. Aphidicolin, an inhibitor of DNA polymerases alpha, delta, and epsilom, inhibits both semiconservative replication and repair synthesis in the extract. Butylphenyl-dGTP also inhibits both replicative and repair synthesis but at a concentration known to inhibit DNA polymerase alpha preferentially rather than delta or epsilon. This suggests that DNA polymerase alpha may function in mismatch repair.     

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.     

Gene conversion in Escherichia coli. Resolution of heteroallelic mismatched nucleotides by co-repair

We have constructed heteroduplex plasmid DNA that is similar in structure to the heteroduplex DNA expected to be produced during genetic recombination of plasmids, and studied its repair after transformation into different Escherichia coli strains. The heteroduplex DNA was constructed using two different parental plasmids, each of which contained a different ten-nucleotide insertion mutation. The effect of different defined states of dam-methylation on repair was also examined. We found that heteroduplex DNA repair occurred prior to the replication of the substrate DNA 60 to 80% of the time, regardless of the state of DNA methylation. Most excision/synthesis tracts covered two markers separated by 1243 base-pairs, and this process has been termed co-repair. The most efficient co-repair pathway was the Dam-instructed repair pathway that required the mutH, mutL, mutS and uvrD gene products and preferentially used the methylated strand as the template for DNA synthesis. If there was no methylation asymmetry, mismatch nucleotide repair occurred with a similar frequency; however, no strand bias was observed. Co-repair of symmetrically methylated heteroduplex DNA required the mutS and uvrD gene products, while repair of unmethylated heteroduplex DNA also required the mutL and mutH gene products.     

A RecG-independent nonconservative branch migration mechanism in Escherichia coli recombination

To gain insight regarding the mechanisms that extend heteroduplex joints in Escherichia coli recombination, we investigated the effect of recG and ruv genotypes on heteroduplex strand polarity in intramolecular recombination products. We also examined the cumulative effect of mutational inactivation of RecG and single-strand-specific exonucleases on recombination proficiency and the role of Chi sites in RecG-independent recombination. All four strands of the two homologs were incorporated into heteroduplex structures in wild-type cells and in ruv mutants. However, in recG mutants heteroduplexes were generated almost exclusively by pairing the invasive 3'-ending strand with its complementary strand. To explain the dependence of strand exchange reciprocity on RecG activity, we propose that alternative mechanisms may extend the heteroduplex joints after homologous pairing: a reciprocal RecG-mediated mechanism and a nonreciprocal mechanism, mediated by RecA and single-strand-specific exonucleases. The cumulative effect of recG and recJ or xonA mutations on recombination proficiency and the inhibitory effect of recJ and xonA activities on heteroduplex formation by the 5'-ending strands are consistent with this proposal.     

Repair of specific base pair mismatches formed during meiotic recombination in the yeast Saccharomyces cerevisiae.

Heteroduplexes formed between DNA strands derived from different homologous chromosomes are an intermediate in meiotic crossing over in the yeast Saccharomyces cerevisiae and other eucaryotes. A heteroduplex formed between wild-type and mutant genes will contain a base pair mismatch; failure to repair this mismatch will lead to postmeiotic segregation (PMS). By analyzing the frequency of PMS for various mutant alleles in the yeast HIS4 gene, we showed that C/C mismatches were inefficiently repaired relative to all other point mismatches. These other mismatches (G/G, G/A, T/T, A/A, T/C, C/A, A/A, and T/G) were repaired with approximately the same efficiency. We found that in spores with unrepaired mismatches in heteroduplexes, the nontranscribed strand of the HIS4 gene was more frequently donated than the transcribed strand. In addition, the direction of repair for certain mismatches was nonrandom.     

Heteroduplex Strand-Specificity in Restriction-Stimulated Recombination by the Rece Pathway of Escherichia Coli

The RecE recombination pathway is active in Escherichia coli recB recC sbcA mutants. To isolate and characterize products and intermediates of RecE-mediated, break-induced, intramolecular recombination, we infected recB recC sbcA mutants, expressing EcoRI endonuclease, with chimeric λ phages that allow EcoRI-mediated release of cloned linear recombination substrates. Substrates with direct terminal repeats recombined to yield a circular product with one copy of the repeated sequence. Some recombinants were heteroallelic for the recombining markers. Markers distant to the break were recovered in the circular product at a higher frequency than markers close to the break. To examine the heteroduplex structures that may have yielded the heteroallelic recombinants, nonreplicative substrates were employed. Some of the nonreplicative recombination products contained heteroduplexes, with a strong bias for paired strands ending 3' at the break. This strand bias in heteroduplex formation is consistent with recombination models that postulate homologous pairing of protruding 3' single-stranded ends.     

Repair and recombination of X-irradiated plasmids in Xenopus laevis oocytes.

Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation. Oocyte-mediated repair of DNA strand breaks was observed with both methods. Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we also demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays. X-irradiation of a circular DNA stimulated its potential to recombine with a homologous linear partner. Recombination products were detected directly by Southern blot hybridization and as bacterial transformant clones expressing two antibiotic resistance markers originally carried separately on the two substrates. The increase in recombination was dependent on X-ray dose. There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination. In summary, oocytes have considerable capacity to repair X-ray-induced damage, and some X-ray lesions stimulate homologous recombination in these cells.     

Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei.

Heteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells.     

Distribution of Exchanges upon Homologous Recombination of Exogenous DNA in Xenopus Laevis Oocytes

Homologous recombination between DNA molecules injected into Xenopus oocyte nuclei was investigated by examining the recovery of information from differentially marked parental sequences. The injected recombination substrate was a linear DNA with terminal direct repeats of 1246 bp; one repeat differed from the other by eight single base-pair substitutions, distributed throughout the region of homology, each of which created or destroyed a restriction enzyme site. Recombination products were recovered and analyzed for their content of the diagnostic sites, either directly by Southern blot-hybridization or after cloning in bacteria. The majority (76%) of the cloned products appeared to be the result of simple exchanges-i.e., there was one sharp transition from sequences derived from one parent to sequences derived from the other. These simple exchanges were concentrated near the ends of the homologous interval and, thus, near the sites of the original molecular ends. Placing marked sites on only one side of the homologous overlap showed that marker recovery was governed largely by the positions of the molecular ends and not by the markers themselves. When a terminal nonhomology was present at one end of the substrate, the yield of recombinants was sharply decreased, but the pattern of exchanges was not affected, suggesting that products from end-blocked substrates arise by the same recombination pathway. Because of considerable evidence supporting a nonconservative, resection-annealing mechanism for recombination in oocytes, we interpret the distribution of exchanges as resulting from long-patch repair of extensive heteroduplex intermediates.     

Endonuclease VII is a key component of the mismatch repair mechanism in bacteriophage T4

In previous papers we described an extra recombination mechanism in T4 phage, which contributed to general recombination only when particular mutations were used as geneticmarkers (high recombination or HR markers), whereas it was practically inactive towards other rIIB mutations (low recombination or LR markers). This marker-dependent recombination pathway was identified as a repair of mismatches in recombination heteroduplexes. We suggested that the first step in this pathway, recognition and incision of the mismatch, is performed by endonuclease VII (endo VII) encoded by the T4 gene 49. In the present paper, we tested this hypothesis in vivo. We used an experimental model system that combines site-specific double-strand breaks with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. We compared recombination of homoallelic HR and LR markers in the S17 and S17 E727 background (amber mutations in the uvsX and in the uvsX and 49 genes, respectively). In S17-crosses, the HR and LR markers retain their respective high-recombination and low-recombination behavior. In S17 E727-crosses, however, the HR and LR markers show no difference in the recombination frequency and both behave as LR markers. We conclude that endo VII is the enzyme that recognizes mismatches in recombinational heteroduplexes and performs their incision. This role for endo VII was suggested previously from biochemical studies, but this is its first in vivo demonstration.     

Highly Mismatched Molecules Resembling Recombination Intermediates Efficiently Transform Mismatch Repair Proficient Escherichia Coli

The ability of related DNAs to undergo recombination decreases with increased sequence divergence. Mismatch repair has been proposed to be a key factor in preventing homeologous recombination; however, the contribution of mismatch repair is not universal. Although mismatch repair has been proposed to act by preventing strand exchange and/or inactivating multiply mismatched heteroduplexes, there has been no systematic study to determine at what step(s) in recombination mismatch repair acts in vivo. Since heteroduplex is a commonly proposed intermediate in many models of recombination, we have investigated the consequences of mismatch repair on plasmids that are multiply mismatched in heteroduplex structures that are similar to those that might arise during recombination. Plasmids containing multiply mismatched regions were transformed into wild-type and Mut(-) Eschericia coli mutants. There was only a 30-40% reduction in transformation of Mut(+) as compared to mutS and mutL strains for DNAs containing an 18% mismatched heteroduplex. The products obtained from mutS hosts differed from those obtained from Mut(+) hosts in that there were many more colonies containing mixtures of two plasmids, due to survival of both strands of the heteroduplex. There were nearly 10 times more recombinants obtained from the mutS as compared to the wild-type host. Based on these results and those from other studies with E. coli and yeast, we propose that the prevention of recombination between highly diverged DNAs may be at step earlier than heteroduplex formation.     

Biased short tract repair of palindromic loop mismatches in mammalian cells.

Mismatch repair of palindromic loops in the presence or absence of single-base mismatches was investigated in wild-type and mismatch-binding defective mutant Chinese hamster ovary cells. Recombination intermediates with a maximum heteroduplex DNA (hDNA) region of 697 bp contained a centrally located, phenotypically silent 12-base palindromic loop mismatch, and/or five single-base mismatches. In wild-type cells, both loops and single-base mismatches were efficiently repaired (80-100%). When no other mismatches were present in hDNA, loops were retained with a 1.6-1.9:1 bias. However, this bias was eliminated when single-base mismatches were present, perhaps because single-base mismatches signal nick-directed repair. In the multiple marker crosses, most repair tracts were long and continuous, with preferential loss of markers in cis to proximal nicks, consistent with nicks directing most repair in this situation. However, approximately 25% of repair tracts were discontinuous as a result of loop-specific repair, or from segregation or short tract repair of single-base mismatches. In mutant cells, single-base mismatches were repaired less frequently, but the loop was still repaired efficiently and with bias toward loop retention, indicating that the defect in these cells does not affect loop-specific repair. Repair tracts in products from mutant cells showed a wide variety of mosaic patterns reflecting short regions of repair and segregation consistent with reduced nick-directed repair. In mutant cells, single-base mismatches were repaired more efficiently in the presence of the loop than in its absence, a likely consequence of corepair initiated at the loop.     

Duplex-duplex interactions catalyzed by recA protein allow strand exchanges to pass double-strand breaks in DNA

Using gapped circular DNA and homologous duplex DNA cut with restriction nucleases, we show that E. coli RecA protein promotes strand exchanges past double-strand breaks. The products of strand exchange are heteroduplex DNA molecules that contain nicks, which can be sealed by DNA ligase, thereby effecting the repair of double-strand breaks in vitro. These results show that RecA protein can promote pairing interactions between homologous DNA molecules at regions where both are duplex. Moreover, pairing leads to strand exchanges and the formation of heteroduplex DNA. In contrast, strand exchanges are unable to pass a double-strand break in the gapped substrate. This apparent paradox is discussed in terms of a model for RecA-DNA interactions in which we propose that each RecA monomer contains two nonequivalent DNA-binding sites.     

Some Features of Base Pair Mismatch and Heterology Repair in Escherichia coli

We have used artificially constructed heteroallelic heteroduplex molecules of bacteriophage lambda DNA to transfect Escherichia coli, and E. coli mutants deficient in various functions involved in the adenine methylation-directed mismatch repair system, MutL, MutS, MutH, and UvrD (MutU). Analysis of the allele content of single infective centers shows that this repair system often acts on several mismatches, separated by as many as 2000 bp, on one of the strands of a heteroduplex molecule. When the methyl-directed mismatch repair system is disabled by mutH or uvrD mutations, localized mismatch repair becomes prominent. This prominent localized repair that can result in separation of very closely linked markers requires the functions MutL and MutS, is independent of adenine methylation, and appears to reflect another mechanism of mismatch repair. Heterology-containing heteroduplex molecules with a deletion in one strand often escape processing. However, when the heterology includes the stem and loop structure of a transposon, Tn10, the transposon is lost.     

Mechanisms of double-strand-break repair during gene targeting in mammalian cells

In the present study, the mechanism of double-strand-break (DSB) repair during gene targeting at the chromosomal immunoglobulin mu-locus in a murine hybridoma was examined. The gene-targeting assay utilized specially designed insertion vectors genetically marked in the region of homology to the chromosomal mu-locus by six diagnostic restriction enzyme site markers. The restriction enzyme markers permitted the contribution of vector-borne and chromosomal mu-sequences in the recombinant product to be determined. The use of the insertion vectors in conjunction with a plating procedure in which individual integrative homologous recombination events were retained for analysis revealed several important features about the mammalian DSB repair process:The presence of the markers within the region of shared homology did not affect the efficiency of gene targeting.In the majority of recombinants, the vector-borne marker proximal to the DSB was absent, being replaced with the corresponding chromosomal restriction enzyme site. This result is consistent with either formation and repair of a vector-borne gap or an "end" bias in mismatch repair of heteroduplex DNA (hDNA) that favored the chromosomal sequence. Formation of hDNA was frequently associated with gene targeting and, in most cases, began approximately 645 bp from the DSB and could encompass a distance of at least 1469 bp.The hDNA was efficiently repaired prior to DNA replication.The repair of adjacent mismatches in hDNA occurred predominantly on the same strand, suggesting the involvement of a long-patch repair mechanism.     

Marker-Dependent Recombination in T4 Bacteriophage. III. Structural Prerequisites for Marker Discrimination

Distance- as well as marker-dependence of genetic recombination of bacteriophage T4 was studied in crosses between rIIB mutants with known base sequences. The notion of a "basic recombination," which is the recombination within distances shorter than hybrid DNA length in the absence of mismatch repair and any marker effects, was substantiated. The basic recombination frequency per base pair can serve as an objective parameter (natural constant) of general recombination reflecting its intensity. Comparative studies of the recombination properties of rIIB mutants with various sequence changes in the mutated sites showed that the main factor determining the probability of mismatch repair in recombination heteroduplexes is the length of a continuous heterologous region. A run of A:T pairs immediately adjoining the mismatch appears to stimulate its repair. In the case of mismatches with DNA strands of unequal length, formed by frameshift mutations, the repair is asymmetric, the longer strand (bulge) being preferentially removed. A pathway for mismatch repair including sequential action of endonuclease VII (gp49)----3'----5' exonuclease (gp43)----DNA polymerase (gp43)----DNA ligase (gp30) was proposed. A possible identity of the recombinational mismatch repair mechanism to that operating to produce mutations via sequence conversion is discussed.