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1.
Y S Weng  J A Nickoloff 《Genetics》1998,148(1):59-70
Double-strand break (DSB) induced gene conversion in Saccharomyces cerevisiae during meiosis and MAT switching is mediated primarily by mismatch repair of heteroduplex DNA (hDNA). We used nontandem ura3 duplications containing palindromic frameshift insertion mutations near an HO nuclease recognition site to test whether mismatch repair also mediates DSB-induced mitotic gene conversion at a non-MAT locus. Palindromic insertions included in hDNA are expected to produce a stem-loop mismatch, escape repair, and segregate to produce a sectored (Ura+/-) colony. If conversion occurs by gap repair, the insertion should be removed on both strands, and converted colonies will not be sectored. For both a 14-bp palindrome, and a 37-bp near-palindrome, approximately 75% of recombinant colonies were sectored, indicating that most DSB-induced mitotic gene conversion involves mismatch repair of hDNA. We also investigated mismatch repair of well-repaired markers flanking an unrepaired palindrome. As seen in previous studies, these additional markers increased loop repair (likely reflecting corepair). Among sectored products, few had additional segregating markers, indicating that the lack of repair at one marker is not associated with inefficient repair at nearby markers. Clear evidence was obtained for low levels of short tract mismatch repair. As seen with full gene conversions, donor alleles in sectored products were not altered. Markers on the same side of the DSB as the palindrome were involved in hDNA less often among sectored products than nonsectored products, but markers on the opposite side of the DSB showed similar hDNA involvement among both product classes. These results can be explained in terms of corepair, and they suggest that mismatch repair on opposite sides of a DSB involves distinct repair tracts.  相似文献   

2.
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.  相似文献   

3.
Repair of Heteroduplex DNA in Xenopus Laevis Oocytes   总被引:2,自引:1,他引:1       下载免费PDF全文
We have hypothesized that the inheritance of heteroallelic markers during recombination of homologous DNAs in Xenopus oocytes is determined by resolution of a heteroduplex intermediate containing multiple single-base mismatches. To test this idea, we prepared synthetic heteroduplexes carrying 8 separate mispairs in vitro and injected them into oocyte nuclei. DNA was recovered and analyzed directly, by Southern blot-hybridization, and indirectly, by cloning individual repair products in bacteria. Mismatch correction was quite efficient in the oocytes; markers on the same strand were commonly co-corrected, indicating a long-patch mechanism; and the distribution of markers was very similar to that obtained by recombination. This supports our interpretation of the recombination outcome in terms of a resection-annealing mechanism. The injected heteroduplexes carried strand breaks (nicks) as a result of their method of preparation. We tested the idea that mismatch correction might be nick-directed by ligating the strands of the heteroduplex substrate to form covalently closed circles. Repair in oocytes was still efficient, and long patches predominated; but the pattern of recovered markers was quite different than with the nicked substrate. This suggests that nicks, when present, do indeed direct repair, but that, in their absence, recognition of specific mismatches governs repair of the ligated heteroduplexes.  相似文献   

4.
Double-strand break (DSB)-induced gene conversion was investigated using plasmid x chromosome (P x C) and chromosomal direct-repeat recombination substrates with markers arranged such that functional (selected) products could not arise by longpatch mismatch repair initiated from the DSB. As seen previously with analogous substrates, these substrates yield products with discontinuous conversion tracts, albeit at low frequency. Most conversion tracts were of minimum length, suggesting that heteroduplex DNA (hDNA) is limiting, or that co-repair imposes selective pressure against products with more extensive hDNA. When functional products can arise by long-patch mismatch repair, the broken allele is converted in nearly all products. In contrast, in the absence of long-patch mismatch repair, unbroken alleles are frequently converted, and we show that such conversion depends on both marker structure (i.e., long palindromic vs. nonpalindromic insertions) and the chromosomal environment of the recombination substrate. We propose that conversion of unbroken alleles is largely a consequence of the segregation of unrepaired markers, and that differences in mismatch repair efficiency underlie the observed effects of marker structure and chromosome environment on allele conversion preference.  相似文献   

5.
Coïc E  Gluck L  Fabre F 《The EMBO journal》2000,19(13):3408-3417
Recombination events between non-identical sequences most often involve heteroduplex DNA intermediates that are subjected to mismatch repair. The well-characterized long-patch mismatch repair process, controlled in eukaryotes by bacterial MutS and MutL orthologs, is the major system involved in repair of mispaired bases. Here we present evidence for an alternative short-patch mismatch repair pathway that operates on a broad spectrum of mismatches. In msh2 mutants lacking the long-patch repair system, sequence analysis of recombination tracts resulting from exchanges between similar but non-identical (homeologous) parental DNAs showed the occurrence of short-patch repair events that can involve <12 nucleotides. Such events were detected both in mitotic and in meiotic recombinants. Confirming the existence of a distinct short-patch repair activity, we found in a recombination assay involving homologous alleles that closely spaced mismatches are repaired independently with high efficiency in cells lacking MSH2 or PMS1. We show that this activity does not depend on genes required for nucleotide excision repair and thus differs from the short-patch mismatch repair described in Schizosaccharomyces pombe.  相似文献   

6.
The repair of 12-, 27-, 62-, and 216-nucleotide unpaired insertion/deletion heterologies has been demonstrated in nuclear extracts of human cells. When present in covalently closed circular heteroduplexes or heteroduplexes containing a single-strand break 3' to the heterology, such structures are subject to a low level repair reaction that occurs with little strand bias. However, the presence of a single-strand break 5' to the insertion/deletion heterology greatly increases the efficiency of rectification and directs repair to the incised DNA strand. Because nick direction of repair is independent of the strand in which a particular heterology is placed, the observed strand bias is not due to asymmetry imposed on the heteroduplex by the extrahelical DNA segment. Strand-specific repair by this system requires ATP and the four dNTPs and is inhibited by aphidicolin. Repair is independent of the mismatch repair proteins MSH2, MSH6, MLH1, and PMS2 and occurs by a mechanism that is distinct from that of the conventional mismatch repair system. Large heterology repair in nuclear extracts of human cells is also independent of the XPF gene product, and extracts of Chinese hamster ovary cells deficient in the ERCC1 and ERCC4 gene products also support the reaction.  相似文献   

7.
Two methods were used in an attempt to increase the efficiency and strand selectivity of methyl-directed mismatch repair of bacteriophage lambda heteroduplexes in E. coli. Previous studies of such repair used lambda DNA that was only partially methylated as the source of methylated chains. Also, transfection was carried out in methylating strains. Either of these factors might have been responsible for the incompleteness of the strand selectivity observed previously. In the first approach to increasing strand selectivity, heteroduplexes were transfected into a host deficient in methylation, but no changes in repair frequencies were observed. In the second approach, heteroduplexes were prepared using DNA that had been highly methylated in vitro with purified DNA adenine methylase as the source of methylated chains. In heteroduplexes having a repairable cI/+ mismatch, strand selectivity was indeed enhanced. In heteroduplexes with one chain highly methylated and the complementary chain unmethylated, the frequency of repair on the unmethylated chain increased to nearly 100%. Heteroduplexes with both chains highly methylated were not repaired at a detectable frequency. Thus, chains highly methylated by DNA adenine methylase were refractory to mismatch repair by this system, regardless of the methylation of the complementary chain. These results support the hypothesis that methyl-directed mismatch repair acts to correct errors of replication, thus lowering the mutation rate.  相似文献   

8.
DNA mismatch repair maintains genomic stability by detecting and correcting mispaired DNA sequences and by signaling cell death when DNA repair fails. The mechanism by which mismatch repair coordinates DNA damage and repair with cell survival or death is not understood, but it suggests the need for regulation. Since the functions of mismatch repair are initiated in the nucleus, we asked whether nuclear transport of MLH1 and PMS2 is limiting for the nuclear localization of MutLalpha (the MLH1-PMS2 dimer). We found that MLH1 and PMS2 have functional nuclear localization signals (NLS) and nuclear export sequences, yet nuclear import depended on their C-terminal dimerization to form MutLalpha. Our studies are consistent with the idea that dimerization of MLH1 and PMS2 regulates nuclear import by unmasking the NLS. Limited nuclear localization of MutLalpha may thus represent a novel mechanism by which cells fine-tune mismatch repair functions. This mechanism may have implications in the pathogenesis of hereditary non-polyposis colon cancer.  相似文献   

9.
Mutations affecting heteroduplex DNA mismatch repair in Streptococcus pneumoniae were localized in two genes, hexA and hexB, by fractionation of restriction fragments carrying mutant alleles. A fragment containing the hexA4 allele was cloned in the S. pneumoniae cloning system, and the hexA+ allele was introduced into the recombinant plasmid by chromosomal facilitation of plasmid transfer. Subcloning localized the functional hexA gene to a 3.5-kilobase segment of the cloned pneumococcal DNA. The product of this gene was shown in Bacillus subtilis minicells to be a polypeptide with an Mr of 86,000. Two mutant alleles of hexA showed partial expression of the repair system when present in multicopy plasmids. A model for mismatch repair, which depends on the interaction of two protein components to recognize the mismatched base pair and excise a segment of DNA between strand breaks surrounding the mismatch, is proposed.  相似文献   

10.
The methyl-directed DNA repair efficiency of a series of M13mp9 frameshift heteroduplexes 1, 2, or 3 unpaired bases was determined by using an in vitro DNA mismatch repair assay. Repair of hemimethylated frameshift heteroduplexes in vitro was directed to the unmethylated strand; was dependent on MutH, MutL, and MutS; and was equally efficient on base insertions and deletions. However, fully methylated frameshift heteroduplexes were resistant to repair, while totally unmethylated substrates were repaired with no strand bias. Hemimethylated 1-, 2-, or 3-base insertion and deletion heteroduplexes were repaired by the methyl-directed mismatch repair pathway as efficiently as the G.T mismatch. These results are consistent with earlier in vivo studies and demonstrate the involvement of methyl-directed DNA repair in the efficient prevention of frameshift mutations.  相似文献   

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