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1.
MutS homologs, identified in nearly all bacteria and eukaryotes, include the bacterial proteins MutS1 and MutS2 and the eukaryotic MutS homologs 1 to 7, and they often are involved in recognition and repair of mismatched bases and small insertion/deletions, thereby limiting illegitimate recombination and spontaneous mutation. To explore the relationship of MutS2 to other MutS homologs, we examined conserved protein domains. Fundamental differences in structure between MutS2 and other MutS homologs suggest that MutS1 and MutS2 diverged early during evolution, with all eukaryotic homologs arising from a MutS1 ancestor. Data from MutS1 crystal structures, biochemical results from MutS2 analyses, and our phylogenetic studies suggest that MutS2 has functions distinct from other members of the MutS family. A mutS2 mutant was constructed in Helicobacter pylori, which lacks mutS1 and mismatch repair genes mutL and mutH. We show that MutS2 plays no role in mismatch or recombinational repair or deletion between direct DNA repeats. In contrast, MutS2 plays a significant role in limiting intergenomic recombination across a range of donor DNA tested. This phenotypic analysis is consistent with the phylogenetic and biochemical data suggesting that MutS1 and MutS2 have divergent functions.  相似文献   

2.
Helicobacter pylori, a human pathogen, is a naturally and constitutively competent bacteria, displaying a high rate of intergenomic recombination. While recombination events are essential for evolution and adaptation of H. pylori to dynamic gastric niches and new hosts, such events should be regulated tightly to maintain genomic integrity. Here, we analyze the role of the nuclease activity of MutS2, a protein that limits recombination during transformation in H. pylori. In previously studied MutS2 proteins, the C‐terminal Smr domain was mapped as the region responsible for its nuclease activity. We report here that deletion of Smr domain does not completely abolish the nuclease activity of HpMutS2. Using bioinformatics analysis and mutagenesis, we identified an additional and novel nuclease motif (LDLK) at the N‐terminus of HpMutS2 unique to Helicobacter and related ε‐proteobacterial species. A single point mutation (D30A) in the LDLK motif and the deletion of Smr domain resulted in ~ 5–10‐fold loss of DNA cleavage ability of HpMutS2. Interestingly, the mutant forms of HpMutS2 wherein the LDLK motif was mutated or the Smr domain was deleted were unable to complement the hyper‐recombination phenotype of a mutS2? strain, suggesting that both nuclease sites are indispensable for an efficient anti‐recombinase activity of HpMutS2.  相似文献   

3.
Multicopy single-stranded DNA (msDNA) molecules consist of single-stranded DNA covalently linked to RNA. In Escherichia coli , such molecules are encoded by genetic elements called retrons. The DNA moieties of msDNAs have characteristic stem-loop structures, and most of these structures contain mismatched base pairs. Previously, we showed that retrons encoding msDNAs with mismatched base pairs are mutagenic when present in multicopy plasmids. In this study we show that such msDNAs, in a similar manner to genetic defects in mismatch repair, increase the frequency of interspecies recombination in matings between Salmonella typhimurium and E. coli . To demonstrate interference with mismatch repair by msDNA, we show that the addition of a plasmid containing the gene for MutS protein suppresses the mutagenic and recombinogenic effects of msDNAs. We also show that in mutS mutants, msDNA does not increase the frequency of either mutations or interspecies recombination. We conclude from these findings that the mutagenic and recombinogenic effects of msDNAs are due to titrating out MutS protein.  相似文献   

4.
DNA recombination events need to be strictly regulated, because an increase in the recombinational frequency causes unfavorable alteration of genetic information. Recent studies revealed the existence of a novel anti-recombination enzyme, MutS2. However, the mechanism by which MutS2 inhibits homologous recombination has been unknown. Previously, we found that Thermus thermophilus MutS2 (ttMutS2) harbors an endonuclease activity and that this activity is confined to the C-terminal domain, whose amino acid sequence is widely conserved in a variety of proteins with unknown function from almost all organisms ranging from bacteria to man. In this study, we determined the crystal structure of the ttMutS2 endonuclease domain at 1.7-angstroms resolution, which resembles the structure of the DNase I-like catalytic domain of Escherichia coli RNase E, a sequence-nonspecific endonuclease. The N-terminal domain of ttMutS2, however, recognized branched DNA structures, including the Holliday junction and D-loop structure, a primary intermediate in homologous recombination. The full-length of ttMutS2 digested the branched DNA structures at the junction. These results indicate that ttMutS2 suppresses homologous recombination through a novel mechanism involving resolution of early intermediates.  相似文献   

5.
Pso2/Snm1 is a member of the beta-CASP metallo-beta-lactamase family of proteins that include the V(D)J recombination factor Artemis. Saccharomyces cerevisiae pso2 mutants are specifically sensitive to agents that induce DNA interstrand cross-links (ICLs). Here we establish a novel overlapping function for PSO2 with MutS mismatch repair factors and the 5'-3' exonuclease Exo1 in the repair of DNA ICLs, which is confined to S phase. Our data demonstrate a requirement for NER and Pso2, or Exo1 and MutS factors, in the processing of ICLs, and this is required prior to the repair of ICL-induced DNA double-strand breaks (DSBs) that form during replication. Using a chromosomally integrated inverted-repeat substrate, we also show that loss of both pso2 and exo1/msh2 reduces spontaneous homologous recombination rates. Therefore, PSO2, EXO1, and MSH2 also appear to have overlapping roles in the processing of some forms of endogenous DNA damage that occur at an irreversibly collapsed replication fork. Significantly, our analysis of ICL repair in cells synchronized for each cell cycle phase has revealed that homologous recombination does not play a major role in the direct repair of ICLs, even in G2, when a suitable template is readily available. Rather, we propose that recombination is primarily involved in the repair of DSBs that arise from the collapse of replication forks at ICLs. These findings have led to considerable clarification of the complex genetic relationship between various ICL repair pathways.  相似文献   

6.
MutS homologues are highly conserved enzymes engaged in DNA mismatch repair (MMR), meiotic recombination and other DNA modifications. Genome sequencing projects have revealed that bacteria and plants possess a MutS homologue, MutS2. MutS2 lacks the mismatch-recognition domain of MutS, but contains an extra C-terminal region called the small MutS-related (Smr) domain. Sequences homologous to the Smr domain are annotated as ‘proteins of unknown function’ in various organisms ranging from bacteria to human. Although recent in vivo studies indicate that MutS2 plays an important role in recombinational events, there had been only limited characterization of the biochemical function of MutS2 and the Smr domain. We previously established that Thermus thermophilus MutS2 (ttMutS2) possesses endonuclease activity. In this study, we report that a Smr-deleted ttMutS2 mutant retains the dimerization, ATPase and DNA-binding activities, but has no endonuclease activity. Furthermore, the Smr domain alone was stable and functional in binding and incising DNA. It is noteworthy that an endonuclease activity is associated with a MutS homologue, which is generally thought to recognize specific DNA structures.  相似文献   

7.
We have characterized the mismatch repair system (MMR) of the highly radiation-resistant type strain of Deinococcus radiodurans, ATCC 13939. We show that the MMR system is functional in this organism, where it participates in ensuring the fidelity of DNA replication and recombination. The system relies on the activity of two key proteins, MutS1 and MutL, which constitute a conserved core involved in mismatch recognition. Inactivation of MutS1 or MutL resulted in a seven-fold increase in the frequency of spontaneous RifR mutagenesis and a ten-fold increase in the efficiency of integration of a donor point-mutation marker during bacterial transformation. Inactivation of the mismatch repair-associated UvrD helicase increased the level of spontaneous mutagenesis, but had no effect on marker integration—suggesting that binding of MutS1 and MutL proteins to a mismatched heteroduplex suffices to inhibit recombination between non identical (homeologous) DNAs. In contrast, inactivation of MutS2, encoded by the second mutS -related gene present in D. radiodurans, had no effect on mutagenesis or recombination. Cells devoid of MutS1 or MutL proteins were as resistant to -rays, mitomycin C and UV-irradiation as wild-type bacteria, suggesting that the mismatch repair system is not essential for the reconstitution of a functional genome after DNA damage.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Baldacci  相似文献   

8.
Sia EA  Kirkpatrick DT 《DNA Repair》2005,4(2):253-261
Six strong homologs of the bacterial MutS DNA mismatch repair (MMR) gene have been identified in the yeast Saccharomyces cerevisiae. With the exception of the MSH1 gene, the involvement of each homolog in DNA repair and recombination during meiosis has been determined previously. Five of the homologs have been demonstrated to act in meiotic DNA repair (MSH2, MSH3, MSH6 and MSH4) and/or meiotic recombination (MSH4 and MSH5). Unfortunately the loss of mitochondrial function that results from deletion of MSH1 disrupts meiotic progression, precluding an analysis of MSH1 function in meiotic DNA repair and recombination. However, the recent identification of two separation-of-function alleles of MSH1 that interfere with protein function but still maintain functional mitochondria allow the meiotic activities of MSH1 to be determined. We show that the G776D and F105A alleles of MSH1 exhibit no defects in meiotic recombination, repair base-base mismatches and large loop mismatches efficiently during meiosis, and have high levels of spore viability. These data indicate that the MSH1 protein, unlike other MutS homologs in yeast, plays no role in DNA repair or recombination during meiosis.  相似文献   

9.
Vsr mediates very short patch repair in Escherichia coli, correcting T/G mismatches caused by deamination of 5-methylcytosine to thymine. MutS and MutL, part of the post-replication mismatch repair system, stimulate VSP repair. In this study, we use a bacterial two-hybrid assay to show that MutL interacts with Vsr. We also show that interaction between Vsr and MutL inhibits the ability of MutL to dimerize, to interact with MutS and MutH and to mediate a previously unknown interaction between MutS and MutH. This inhibition may explain why high levels of Vsr are mutagenic in vivo. In addition, we show that the Mut fusion proteins are repair proficient in the bacterial two-hybrid assay, making it possible to study their interactions in various genetic backgrounds, or in the presence of DNA damaging agents.  相似文献   

10.
Antony E  Hingorani MM 《Biochemistry》2004,43(41):13115-13128
Prokaryotic MutS and eukaryotic Msh proteins recognize base pair mismatches and insertions or deletions in DNA and initiate mismatch repair. These proteins function as dimers (and perhaps higher order oligomers) and possess an ATPase activity that is essential for DNA repair. Previous studies of Escherichia coli MutS and eukaryotic Msh2-Msh6 proteins have revealed asymmetry within the dimer with respect to both DNA binding and ATPase activities. We have found the Thermus aquaticus MutS protein amenable to detailed investigation of the nature and role of this asymmetry. Here, we show that (a) in a MutS dimer one subunit (S1) binds nucleotide with high affinity and the other (S2) with 10-fold weaker affinity, (b) S1 hydrolyzes ATP rapidly while S2 hydrolyzes ATP at a 30-50-fold slower rate, (c) mismatched DNA binding to MutS inhibits ATP hydrolysis at S1 but slow hydrolysis continues at S2, and (d) interaction between mismatched DNA and MutS is weakened when both subunits are occupied by ATP but remains stable when S1 is occupied by ATP and S2 by ADP. These results reveal key MutS species in the ATPase pathway; S1(ADP)-S2(ATP) is formed preferentially in the absence of DNA or in the presence of fully matched DNA, while S1(ATP)-S2(ATP) and S1(ATP)-S2(ADP) are formed preferentially in the presence of mismatched DNA. These MutS species exhibit differences in interaction with mismatched DNA that are likely important for the mechanism of MutS action in DNA repair.  相似文献   

11.
Functional analysis of the Arabidopsis thaliana mismatch repair gene MSH2.   总被引:2,自引:0,他引:2  
J Adé  Y Haffani  F J Beizile 《Génome》2001,44(4):651-657
The Arabidopsis thaliana MSH2 (AtMSH2) gene encodes a protein that belongs to a family of highly conserved proteins (MutS homologues (MSH)) involved in DNA mismatch repair. Sequence analysis strongly suggests that this single copy gene is indeed a homologue of MSH2, a gene known to play a central role in eukaryotic mismatch repair. In this report, we show that the AtMSH2 protein has functional attributes characteristic of previously described mismatch repair proteins. First, over-expression of this protein in Escherichia coli leads to a mutator phenotype similar to that reported previously for known functional homologues. Second, gel retardation assays revealed that the AtMSH2 protein has a 10-fold greater affinity for DNA containing a single pair of mismatched nucleotides versus perfectly matched DNA. These results provide experimental evidence that AtMSH2 is indeed a functional homologue of MutS.  相似文献   

12.
Mismatched base-pairs, which are caused by either DNA replication errors, DNA damage or genetic recombination, are repaired by the mismatch-repair system. The MutS protein, a component of the mismatch-repair system, recognizes mismatched base-pairs in DNA, and its DNA-binding activity is affected by ATP and ADP. Here, we show that the MutS protein from Thermus thermophilus HB8 can have three different conformations in solution, based on direct observations made by small-angle X-ray scattering. The conformation of MutS in solution is drastically influenced by the presence of ADP and ATP; the ATP-bound form has the most compact conformation, the ADP-bound form the most stretched, and the nucleotide-free form has a conformation intermediate between the two. Based on these findings, we conclude that the DNA-binding activity of MutS may depend on conformational changes triggered by both the binding and hydrolysis of ATP.  相似文献   

13.
DNA mismatch repair (MMR) is initiated when the MutS protein recognizes damaged DNA. Crystal structures of MutS bound to mispaired and unpaired DNA show how MutS distinguishes damaged from undamaged DNA and explain how a broad variety of DNA mismatch lesions can be detected. The structures suggest mechanisms for the ATP-induced structural regulation of multistep DNA repair processes.  相似文献   

14.
DNA mismatch repair in Escherichia coli has been shown to be involved in two distinct processes: mutation avoidance, which removes potential mutations arising as replication errors, and antirecombination which prevents recombination between related, but not identical (homeologous), DNA sequences. We show that cells with the mutSΔ800 mutation (which removes the C-terminal 53 amino acids of MutS) on a multicopy plasmid are proficient for mutation avoidance. In interspecies genetic crosses, however, recipients with the mutSΔ800 mutation show increased recombination by up to 280-fold relative to mutS+. The MutSΔ800 protein binds to O6-methylguanine mismatches but not to intrastrand platinated GG cross-links, explaining why dam bacteria with the mutSΔ800 mutation are resistant to cisplatin, but not MNNG, toxicity. The results indicate that the C-terminal end of MutS is necessary for antirecombination and cisplatin sensitization, but less significant for mutation avoidance. The inability of MutSΔ800 to form tetramers may indicate that these are the active form of MutS.  相似文献   

15.
In Escherichia coli, errors in newly-replicated DNA, such as the incorporation of a nucleotide with a mis-paired base or an accidental insertion or deletion of nucleotides, are corrected by a methyl-directed mismatch repair (MMR) pathway. While the enzymology of MMR has long been established, many fundamental aspects of its mechanisms remain elusive, such as the structures, compositions, and orientations of complexes of MutS, MutL, and MutH as they initiate repair. Using atomic force microscopy, we—for the first time—record the structures and locations of individual complexes of MutS, MutL and MutH bound to DNA molecules during the initial stages of mismatch repair. This technique reveals a number of striking and unexpected structures, such as the growth and disassembly of large multimeric complexes at mismatched sites, complexes of MutS and MutL anchoring latent MutH onto hemi-methylated d(GATC) sites or bound themselves at nicks in the DNA, and complexes directly bridging mismatched and hemi-methylated d(GATC) sites by looping the DNA. The observations from these single-molecule studies provide new opportunities to resolve some of the long-standing controversies in the field and underscore the dynamic heterogeneity and versatility of MutSLH complexes in the repair process.  相似文献   

16.
The genus Acinetobacter encompasses a heterogeneous group of bacteria that are ubiquitous in the natural environment due in part to their ability to adapt genetically to novel challenges. Acinetobacter sp. strain ADP1 (also known as strain BD413) is naturally transformable and takes up DNA from any source. Donor DNA can be integrated into the chromosome by recombination provided it possesses sufficient levels of nucleotide sequence identity to the recipient's DNA. In other bacteria, the requirement for sequence identity during recombination is partly due to the actions of the mismatch repair system, a key component of which, MutS, recognizes mismatched bases in heteroduplex DNA and, along with MutL, blocks strand exchange. We have cloned mutS from strain ADP1 and examined its roles in preventing recombination between divergent DNA and in the repair of spontaneous replication errors. Inactivation of mutS resulted in 3- to 17-fold increases in transformation efficiencies with donor sequences that were 8 to 20% divergent relative to the strain ADP1. Strains lacking MutS exhibited increased spontaneous mutation frequencies, and reversion assays demonstrated that MutS preferentially recognized transition mismatches while having little effect on the repair of transversion mismatches. Inactivation of mutS also abolished the marker-specific variations in transforming efficiency seen in mutS(+) recipients where transition and frameshift alleles transformed at eightfold lower frequencies than transversions or large deletions. Comparison of the MutS homologs from five individual Acinetobacter strains with those of other gram-negative bacteria revealed that a number of unique indels are conserved among the Acinetobacter amino acid sequences.  相似文献   

17.
Mismatch repair (MMR) corrects DNA polymerase errors occurring during genome replication. MMR is critical for genome maintenance, and its loss increases mutation rates several hundred fold. Recent work has shown that the interaction between the mismatch recognition protein MutS and the replication processivity clamp is important for MMR in Bacillus subtilis. To further understand how MMR is coupled to DNA replication, we examined the subcellular localization of MMR and DNA replication proteins fused to green fluorescent protein (GFP) in live cells, following an increase in DNA replication errors. We demonstrate that foci of the essential DNA polymerase DnaE-GFP decrease following mismatch incorporation and that loss of DnaE-GFP foci requires MutS. Furthermore, we show that MutS and MutL bind DnaE in vitro, suggesting that DnaE is coupled to repair. We also found that DnaE-GFP foci decrease in vivo following a DNA damage-independent arrest of DNA synthesis showing that loss of DnaE-GFP foci is caused by perturbations to DNA replication. We propose that MutS directly contacts the DNA replication machinery, causing a dynamic change in the organization of DnaE at the replication fork during MMR. Our results establish a striking and intimate connection between MMR and the replicating DNA polymerase complex in vivo.  相似文献   

18.
The MutS1 protein recognizes unpaired bases and initiates mismatch repair, which are essential for high-fidelity DNA replication. The homologous MutS2 protein does not contribute to mismatch repair, but suppresses homologous recombination. MutS2 lacks the damage-recognition domain of MutS1, but contains an additional C-terminal extension: the small MutS-related (Smr) domain. This domain, which is present in both prokaryotes and eukaryotes, has previously been reported to bind to DNA and to possess nicking endonuclease activity. We determine here the solution structure of the functionally active Smr domain of the Bcl3-binding protein (also known as Nedd4-binding protein 2), a protein with unknown function that lacks other domains present in MutS proteins. The Smr domain adopts a two-layer α-β sandwich fold, which has a structural similarity to the C-terminal domain of IF3, the R3H domain, and the N-terminal domain of DNase I. The most conserved residues are located in three loops that form a contiguous, exposed, and positively charged surface with distinct sequence identity for prokaryotic and eukaryotic Smr domains. NMR titration experiments and DNA binding studies using Bcl3-binding protein-Smr domain mutants suggested that these most conserved loop regions participate in DNA binding to single-stranded/double-stranded DNA junctions. Based on the observed DNA-binding-induced multimerization, the structural similarity with both subdomains of DNase I, and the experimentally identified DNA-binding surface, we propose a model for DNA recognition by the Smr domain.  相似文献   

19.
Werner syndrome (WS) is a severe recessive disorder characterized by premature aging, cancer predisposition and genomic instability. The gene mutated in WS encodes a bi-functional enzyme called WRN that acts as a RecQ-type DNA helicase and a 3′-5′ exonuclease, but its exact role in DNA metabolism is poorly understood. Here we show that WRN physically interacts with the MSH2/MSH6 (MutSα), MSH2/MSH3 (MutSβ) and MLH1/PMS2 (MutLα) heterodimers that are involved in the initiation of mismatch repair (MMR) and the rejection of homeologous recombination. MutSα and MutSβ can strongly stimulate the helicase activity of WRN specifically on forked DNA structures with a 3′-single-stranded arm. The stimulatory effect of MutSα on WRN-mediated unwinding is enhanced by a G/T mismatch in the DNA duplex ahead of the fork. The MutLα protein known to bind to the MutS α–heteroduplex complexes has no effect on WRN-mediated DNA unwinding stimulated by MutSα, nor does it affect DNA unwinding by WRN alone. Our data are consistent with results of genetic experiments in yeast suggesting that MMR factors act in conjunction with a RecQ-type helicase to reject recombination between divergent sequences.  相似文献   

20.
When DNA mismatch repair fails, the result is a mutator phenotype, which can lead to cancer in humans. Functional repair is dependent on the recognition of mismatches by a dimeric MutS protein, a homodimer in bacteria but a heterodimer in humans. Recent crystal structures of Thermus aquaticus and Escherichia coli MutS have revealed the structural heterodimeric nature of the bacterial proteins and provide new insights into their complicated ATP-dependent repair mechanism.  相似文献   

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