Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2–Msh6 mismatch repair protein

Antony, Edwin; Khubchandani, Sapna; Chen, Siying; Hingorani, Manju

doi:10.1016/j.dnarep.2005.08.016

Cited by 43 publications

(46 citation statements)

References 37 publications

(83 reference statements)

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“…Once Msh2-Msh6 is bound to base-paired DNA, ATP binding causes Msh2-Msh6 to directly dissociate from DNA (21). In contrast, when Msh2-Msh6 is bound to a mispair, ATP hydrolysis at the Msh6 site is inhibited, which then favors binding of ATP at the Msh2 site (32,37,38). A similar dual ATPbound state can be achieved with ATP␥S (21,32).…”

Section: Dna Mismatch Repair (Mmr)mentioning

confidence: 72%

Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Hargreaves

Shell

Mazur

et al. 2010

Journal of Biological Chemistry

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DNA mismatch repair (MMR)2 recognizes and corrects mispaired nucleotides that arise in DNA as a result of errors during DNA replication and chemical damage to DNA and DNA precursors and during the formation of heteroduplex recombination intermediates (1-5). A key early step in MMR is the recognition of the mispaired base in the DNA. In bacteria, mispaired bases are recognized by the MutS homodimer (2, 3, 6 -9). Eukaryotes possess multiple MutS homologs that form two heterodimers, Msh2-Msh6 and Msh2-Msh3, which have distinct but partially overlapping mispair-binding specificities (4, 10 -14). Binding of both mispaired DNA and ATP converts MutS, as well as Msh2-Msh6 and Msh2-Msh3, into a form that is capable of recruiting other MMR proteins and triggering downstream events in MMR (15-23).Structures of truncated forms of bacterial MutS (2, 6) and human Msh2-Msh6 (24) in complex with DNA mispairs have been determined by x-ray crystallography. MutS homodimers bind mispaired DNA and form asymmetric rings around the DNA in which only one subunit contacts the mispaired base (2,6,7,25,26). This basic structure is conserved in Msh2-Msh6, with Msh6 being the mispair-contacting subunit (24,(27)(28)(29). Consistent with known structures, challenging the mispairbound forms of MutS, Msh2-Msh6, and Msh2-Msh3 with ATP converts them to a sliding clamp form that slides freely along the DNA but is trapped on an end-blocked DNA (15,16,21,30,31). In contrast, the base pair-bound form undergoes direct dissociation from the DNA when challenged with ATP (21, 32). Taken together, these observations suggest that mispair binding may license an ATP binding-dependent conformational change that results in conversion to the sliding form. Furthermore, only the mispair-bound forms of MutS and Msh2-Msh6 form ATP-dependent ternary complexes with MutL and Mlh1-Pms1, respectively, which facilitate subsequent steps in MMR (15-17, 21, 23, 33-35). Attempts to understand the ATPbound conformations of these complexes by soaking ATP and ATP analogs into the MutS-mispair crystals have thus far failed to induce conformational changes expected from the biochemical characterization of these complexes, possibly because of restraints placed on the proteins by the crystal lattice (2, 6, 24 -26). Thus, the nature of the ATP binding-induced conformational changes that link mispair recognition with conversion to the sliding form and/or the form that is competent for ternary complex formation is presently unknown.The interactions between the bacterial and eukaryotic mispair-binding proteins and ATP are probably best understood for the Msh2-Msh6 complex (32). In the absence of DNA, the Msh6 nucleotide-binding site has high affinity for ATP and low affinity for ADP, whereas the Msh2 nucleotide-binding site has lower affinity for ATP and higher affinity for ADP. ATP binding at the Msh6 nucleotide-binding site results in reduced affinity for ADP in the Msh2 nucleotide-binding site. In the absence of mispair binding, the ATP in the Msh6 nucleotide-binding site is rapid...

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Section: Dna Mismatch Repair (Mmr)mentioning

confidence: 72%

Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Hargreaves

Shell

Mazur

et al. 2010

Journal of Biological Chemistry

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“…In the msh6 strain, levels of both contractions and sectored colonies dramatically dropped, and were not statistically different from those In order to determine if MSH2 ATPase activity was required for heteroduplex formation, we introduced a point mutation in the MSH2 Walker B motif (Glu768->Ala768). This mutation does not affect heterodimerization nor the ability to bind mismatched DNA but dramatically reduces ATPase activity and results in mutation rates similar to the null allele [52,53]. The msh2-E768A gene was placed under the control of the same doxycyclin-regulated (TetO) 7 promoter and expressed as previously.…”

Section: Muts Complexes Promote Cag/ctg Heteroduplex Formation or Stamentioning

confidence: 99%

Replication stalling and heteroduplex formation within CAG/CTG trinucleotide repeats by mismatch repair

et al. 2016

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2 Highlights-CAG/CTG triplet repeats block replication in both orientations on a yeast chromosome -Replication fork stalling depends on mismatch repair integrity -Msh2p is enriched at CAG/CTG triplet repeats in both orientations -MSH2 overexpression favors or stabilizes the formation of heteroduplex molecules 3 AbstractTrinucleotide repeat expansions are responsible for at least two dozen neurological disorders.Mechanisms leading to these large expansions of repeated DNA are still poorly understood. It was proposed that transient stalling of the replication fork by the repeat tract might trigger slippage of the newly-synthesized strand over its template, leading to expansions or contractions of the triplet repeat. However, such mechanism was never formally proven. Here we show that replication fork pausing and CAG/CTG trinucleotide repeat instability are not linked, stable and unstable repeats exhibiting the same propensity to stall replication forks when integrated in a yeast natural chromosome. We found that replication fork stalling was dependent on the integrity of the mismatch-repair system, especially the Msh2p-Msh6p complex, suggesting that direct interaction of MMR proteins with secondary structures formed by trinucleotide repeats in vivo, triggers replication fork pauses. We also show by chromatin immunoprecipitation that Msh2p is enriched at trinucleotide repeat tracts, in both stable and unstable orientations, this enrichment being dependent on MSH3 and MSH6.Finally, we show that overexpressing MSH2 favors the formation of heteroduplex regions, leading to an increase in contractions and expansions of CAG/CTG repeat tracts during replication, these heteroduplexes being dependent on both MSH3 and MSH6. These heteroduplex regions were not detected when a mutant msh2-E768A gene in which the ATPase domain was mutated was overexpressed. Our results unravel two new roles for mismatch-repair proteins: stabilization of heteroduplex regions and transient blocking of replication forks passing through such repeats. Both roles may involve direct interactions between MMR proteins and secondary structures formed by trinucleotide repeat tracts, although indirect interactions may not be formally excluded.

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“…Recent studies have revealed asymmetry in the ATPase activity of the two subunits in the MutS dimer, 14,15,[18][19][20][21][22] which correlates with asymmetry in their DNA-binding activity; only one subunit provides Phe and Glu residues for base-specific interactions with the mismatch. 7,8,23 One subunit (S1 in MutS, Msh6 in Msh2-Msh6) binds ATP with high affinity and hydrolyzes it rapidly when MutS is alone or with matched DNA, while the other subunit (S2 in MutS, Msh2 in Msh2-Msh6) appears to hydrolyze ATP slowly.…”

Section: Introductionmentioning

confidence: 99%

“…It is clear that the ATPase activities of the two subunits are linked, but exactly how ATP binding, hydrolysis, and product release are coordinated between the two is still under investigation. 18,20 The subunit asymmetry potentially increases the complexity of coupling between the DNA-binding and ATPase activities of MutS, as there are now up to nine possible nucleotide-bound and nucleotide-free forms of the dimer whose formation and decay could influence its actions on DNA during mismatch repair.…”

Section: Introductionmentioning

confidence: 99%

The Effects of Nucleotides on MutS-DNA Binding Kinetics Clarify the Role of MutS ATPase Activity in Mismatch Repair

Jacobs-Palmer

Hingorani

2007

Journal of Molecular Biology

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MutS protein initiates mismatch repair with recognition of a non-WatsonCrick base-pair or base insertion/deletion site in DNA, and its interactions with DNA are modulated by ATPase activity. Here, we present a kinetic analysis of these interactions, including the effects of ATP binding and hydrolysis, reported directly from the mismatch site by 2-aminopurine fluorescence. When free of nucleotides, the Thermus aquaticus MutS dimer binds a mismatch rapidly (k ON = 3 × 10 6 M −1 s −1 ) and forms a stable complex with a half-life of 10 s (k OFF = 0.07 s −1 ). When one or both nucleotide-binding sites on the MutS•mismatch complex are occupied by ATP, the complex remains fairly stable, with a half-life of 5-7 s (k OFF = 0.1-0.14 s −1 ), although MutS ATP becomes incapable of (re-)binding the mismatch. When one or both nucleotide-binding sites on the MutS dimer are occupied by ADP, the MutS•mismatch complex forms rapidly (k ON = 7.3 × 10 6 M −1 s −1 ) and also dissociates rapidly, with a half-life of 0.4 s (k OFF = 1.7 s −1 ). Integration of these MutS DNA-binding kinetics with previously described ATPase kinetics reveals that: (a) in the absence of a mismatch, MutS in the ADPbound form engages in highly dynamic interactions with DNA, perhaps probing base-pairs for errors; (b) in the presence of a mismatch, MutS stabilized in the ATP-bound form releases the mismatch slowly, perhaps allowing for onsite interactions with downstream repair proteins; (c) ATPbound MutS then moves off the mismatch, perhaps as a mobile clamp facilitating repair reactions at distant sites on DNA, until ATP is hydrolyzed (or dissociates) and the protein turns over.

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Contribution of Msh2 and Msh6 subunits to the asymmetric ATPase and DNA mismatch binding activities of Saccharomyces cerevisiae Msh2–Msh6 mismatch repair protein

Cited by 43 publications

References 37 publications

Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Replication stalling and heteroduplex formation within CAG/CTG trinucleotide repeats by mismatch repair

The Effects of Nucleotides on MutS-DNA Binding Kinetics Clarify the Role of MutS ATPase Activity in Mismatch Repair

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