New insights into how MutS separates its function in the regulation of the Pol IV access to replication sites from that in the conserved MMR pathway (original) (raw)
Roles for mismatch repair factors in regulating genetic recombination
Molecular and cellular biology, 2000
Mismatch repair (MMR) systems are evolutionarily conserved and play a primary role in mutation avoidance by removing base-base and small insertion-deletion mismatches that arise during DNA replication (31). In addition, MMR factors are required for the repair of mismatches in heteroduplex DNA (hDNA) that form as a result of sequence heterologies between recombining sequences . MMR also acts to inhibit recombination between moderately divergent (homeologous) sequences . The roles of MMR during recombination are believed to reflect the interaction of MMR factors with mismatches that arise in hDNA or possibly with other structures such as Holliday junctions (2, 33). The full range of effects that MMR can exert on mitotic and meiotic recombination have been discussed elsewhere (11) and will only be summarized briefly here. The purpose of this review is to highlight recent results that have furthered our understanding of interactions between MMR factors and mitotic recombination intermediates.
Mismatch repair and DNA damage signalling
DNA Repair, 2004
Postreplicative mismatch repair (MMR) increases the fidelity of DNA replication by up to three orders of magnitude, through correcting DNA polymerase errors that escaped proofreading. MMR also controls homologous recombination (HR) by aborting strand exchange between divergent DNA sequences. In recent years, MMR has also been implicated in the response of mammalian cells to DNA damaging agents. Thus, MMR-deficient cells were shown to be around 100-fold more resistant to killing by methylating agents of the S N 1type than cells with functional MMR. In the case of cisplatin, the sensitivity difference was lower, typically two-to three-fold, but was observed in all matched MMR-proficient and -deficient cell pairs. More controversial is the role of MMR in cellular response to other DNA damaging agents, such as ionizing radiation (IR), topoisomerase poisons, antimetabolites, UV radiation and DNA intercalators. The MMR-dependent DNA damage signalling pathways activated by the above agents are also ill-defined. To date, signalling cascades involving the Ataxia telangiectasia mutated (ATM), ATM-and Rad3-related (ATR), as well as the stress-activated kinases JNK/SAPK and p38␣ have been linked with methylating agent and 6-thioguanine (TG) treatments, while cisplatin damage was reported to activate the c-Abl and JNK/SAPK kinases in MMR-dependent manner. MMR defects are found in several different cancer types, both familiar and sporadic, and it is possible that the involvement of the MMR system in DNA damage signalling play an important role in transformation. The scope of this article is to provide a brief overview of the recent literature on this subject and to raise questions that could be addressed in future studies.
MutSΒ exceeds MutSα in dinucleotide loop repair
British Journal of Cancer, 2010
The target substrates of DNA mismatch recognising factors MutSalpha (MSH2+MSH6) and MutSbeta (MSH2+MSH3) have already been widely researched. However, the extent of their functional redundancy and clinical substance remains unclear. Mismatch repair (MMR)-deficient tumours are strongly associated with microsatellite instability (MSI) and the degree and type of MSI seem to be dependent on the MMR gene affected, and is linked to its substrate specificities. Deficiency in MSH2 and MSH6 is associated with both mononucleotide and dinucleotide repeat instability. Although no pathogenic MSH3 mutations have been reported, its deficiency is also suggested to cause low dinucleotide repeat instability. To assess the substrate specificities and functionality of MutSalpha and MutSbeta we performed an in vitro MMR assay using three substrate constructs, GT mismatch, 1 and 2 nucleotide insertion/deletion loops (IDLs) in three different cell lines. Our results show that though MutSalpha alone seems to be responsible for GT and IDL1 repair, MutSalpha and MutSbeta indeed have functional redundancy in IDL2 repair and in contrast with earlier studies, MutSbeta seems to exceed MutSalpha. The finding is clinically relevant because the strong role of MutSbeta in IDL2 repair indicates MSH3 deficiency in tumours with low dinucleotide and no mononucleotide repeat instability.
Unexpected moves: a conformational change in MutSα enables high-affinity DNA mismatch binding
Nucleic Acids Research
The DNA mismatch repair protein MutSα recognizes wrongly incorporated DNA bases and initiates their correction during DNA replication. Dysfunctions in mismatch repair lead to a predisposition to cancer. Here, we study the homozygous mutation V63E in MSH2 that was found in the germline of a patient with suspected constitutional mismatch repair deficiency syndrome who developed colorectal cancer before the age of 30. Characterization of the mutant in mouse models, as well as slippage and repair assays, shows a mildly pathogenic phenotype. Using cryogenic electron microscopy and surface plasmon resonance, we explored the mechanistic effect of this mutation on MutSα function. We discovered that V63E disrupts a previously unappreciated interface between the mismatch binding domains (MBDs) of MSH2 and MSH6 and leads to reduced DNA binding. Our research identifies this interface as a ‘safety lock’ that ensures high-affinity DNA binding to increase replication fidelity. Our mechanistic mode...
The crystal structure of DNA mismatch repair protein MutS binding to a G⋅T mismatch
DNA mismatch repair ensures genomic integrity on DNA replication. Recognition of a DNA mismatch by a dimeric MutS protein initiates a cascade of reactions and results in repair of the newly synthesized strand; however, details of the molecular mechanism remain controversial. Here we present the crystal structure at 2.2 A ˚ of MutS from Escherichia coli bound to a G⋅T mismatch. The two MutS monomers have different conformations and form a heterodimer at the structural level. Only one monomer recognizes the mismatch specifically and has ADP bound. Mismatch recognition occurs by extensive minor groove interactions causing unusual base pairing and kinking of the DNA. Nonspecific major groove DNA-binding domains from both monomers embrace the DNA in a clamp-like structure. The interleaved nucleotide-binding sites are located far from the DNA. Mutations in human MutS (MSH2/ MSH6) that lead to hereditary predisposition for cancer, such as hereditary non-polyposis colorectal cancer, can be mapped to this crystal structure.
Cell, 2011
DNA mismatch repair (MMR) increases replication fidelity by eliminating mispaired bases resulting from replication errors. In Saccharomyces cerevisiae, mispairs are primarily detected by the Msh2-Msh6 complex and corrected following recruitment of the Mlh1-Pms1 complex. Here, we visualized functional fluorescent versions of Msh2-Msh6 and Mlh1-Pms1 in living cells. We found that the Msh2-Msh6 complex is an S phase component of replication centers independent of mispaired bases; this localized pool accounted for 10%-15% of MMR in wildtype cells but was essential for MMR in the absence of Exo1. Unexpectedly, Mlh1-Pms1 formed nuclear foci that, although dependent on Msh2-Msh6 for formation, rarely colocalized with Msh2-Msh6 replication-associated foci. Mlh1-Pms1 foci increased when the number of mispaired bases was increased; in contrast, Msh2-Msh6 foci were unaffected. These findings suggest the presence of replication machinery-coupled and -independent pathways for mispair recognition by Msh2-Msh6, which direct formation of superstoichiometric Mlh1-Pms1 foci that represent sites of active MMR.
Mutations affecting a putative MutLα endonuclease motif impact multiple mismatch repair functions
DNA Repair, 2007
Mutations in DNA mismatch repair (MMR) lead to increased mutation rates and higher recombination between similar, but not identical sequences, as well as resistance to certain DNA methylating agents. Recently, a component of human MMR machinery, MutLα, has been shown to display a latent endonuclease activity. The endonuclease active site appears to include a conserved motif, DQHA(X) 2 E(X) 4 E, within the COOH-terminus of human PMS2. Substitution of the glutamic acid residue (E705) abolished the endonuclease activity and mismatch-dependent excision in vitro. Previously, we showed that the PMS2-E705K mutation and the corresponding mutation in Saccharomyces cerevisiae were both recessive loss of function alleles for mutation avoidance in vivo. Here, we show that mutations impacting this endonuclease motif also significantly affect MMRdependent suppression of homeologous recombination in yeast and responses to S n 1-type methylating agents in both yeast and mammalian cells. Thus, our in vivo results suggest that the endonuclease activity of MutLα is important not only in MMR-dependent mutation avoidance but also for recombination and damage response functions.
Mechanism of MutS Searching for DNA Mismatches and Signaling Repair
Journal of Biological Chemistry, 2008
DNA mismatch repair is initiated by the recognition of mismatches by MutS proteins. The mechanism by which MutS searches for and recognizes mismatches and subsequently signals repair remains poorly understood. We used single-molecule analyses of atomic force microscopy images of MutS-DNA complexes, coupled with biochemical assays, to determine the distributions of conformational states, the DNA binding affinities, and the ATPase activities of wild type and two mutants of MutS, with alanine substitutions in the conserved Phe-Xaa-Glu mismatch recognition motif. We find that on homoduplex DNA, the conserved Glu, but not the Phe, facilitates MutS-induced DNA bending, whereas at mismatches, both Phe and Glu promote the formation of an unbent conformation. The data reveal an unusual role for the Phe residue in that it promotes the unbending, not bending, of DNA at mismatch sites. In addition, formation of the specific unbent MutS-DNA conformation at mismatches appears to be required for the inhibition of ATP hydrolysis by MutS that signals initiation of repair. These results provide a structural explanation for the mechanism by which MutS searches for and recognizes mismatches and for the observed phenotypes of mutants with substitutions in the Phe-Xaa-Glu motif.
Mutations in the MutS interaction interface of MLH1 can abolish DNA mismatch repair
Nucleic Acids Research, 2006
MutLa, a heterodimer of MLH1 and PMS2, plays a central role in human DNA mismatch repair. It interacts ATP-dependently with the mismatch detector MutSa and assembles and controls further repair enzymes. We tested if the interaction of MutLa with DNA-bound MutSa is impaired by cancer-associated mutations in MLH1, and identified one mutation (Ala128Pro) which abolished interaction as well as mismatch repair activity. Further examinations revealed three more residues whose mutation interfered with interaction. Homology modelling of MLH1 showed that all residues clustered in a small accessible surface patch, suggesting that the major interaction interface of MutLa for MutSa is located on the edge of an extensive b-sheet that backs the MLH1 ATP binding pocket. Bioinformatic analysis confirmed that this patch corresponds to a conserved potential protein-protein interaction interface which is present in both human MLH1 and its E.coli homologue MutL. MutL could be sitespecifically crosslinked to MutS from this patch, confirming that the bacterial MutL-MutS complex is established by the corresponding interface in MutL. This is the first study that identifies the conserved major MutLa-MutSa interaction interface in MLH1 and demonstrates that mutations in this interface can affect interaction and mismatch repair, and thereby can also contribute to cancer development.
Endonucleolytic Function of MutLα in Human Mismatch Repair
Cell, 2006
Half of hereditary nonpolyposis colon cancer kindreds harbor mutations that inactivate MutLa (MLH1PMS2 heterodimer). MutLa is required for mismatch repair, but its function in this process is unclear. We show that human MutLa is a latent endonuclease that is activated in a mismatch-, MutSa-, RFC-, PCNA-, and ATP-dependent manner. Incision of a nicked mismatch-containing DNA heteroduplex by this four-protein system is strongly biased to the nicked strand. A mismatch-containing DNA segment spanned by two strand breaks is removed by the 5 0-to-3 0 activity of MutSaactivated exonuclease I. The probable endonuclease active site has been localized to a PMS2 DQHA(X) 2 E(X) 4 E motif. This motif is conserved in eukaryotic PMS2 homologs and in MutL proteins from a number of bacterial species but is lacking in MutL proteins from bacteria that rely on d(GATC) methylation for strand discrimination in mismatch repair. Therefore, the mode of excision initiation may differ in these organisms.