Stoichiometry of MutS and MutL at unrepaired mismatches in vivo suggests a mechanism of repair

Elez, Marina; Radman, Miroslav; Matić, Ivan

doi:10.1093/nar/gkr1298

Cited by 43 publications

(53 citation statements)

References 39 publications

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“…In addition, the total number of proteins in the complex is similar to the number of proteins in complexes of yeast MutSα-MutLα detected by surface plasmon resonance (23). The observed excess of MutL over MutS contrasts with the proposed 1:1 stoichiometry in MutS-MutL sliding clamps (16,24), but agrees with in vivo studies in E. coli and yeast, where repair foci contain more MutL than MutS proteins (18,22), and with early DNA footprinting studies indicating complexes containing multiple MutS and MutL proteins at the mismatch (3,25). Consistent with the latter observation, additional crosslinking experiments using unlabeled MutL, Alexa 555-tagged MutS and the Cy5-T-bulge-DNA revealed FRET in all complexes, confirming their presence near the mismatch, consistent with our dynamic experiments with uncrosslinked proteins (Fig.…”

Section: Resultssupporting

confidence: 82%

“…2D). Rather than a sliding MutSMutL clamp model, our findings suggest a model in which MutL flanks MutS at the mismatch, as first suggested by Modrich and coworkers (6, 7) and more recently by other investigators (18,22).…”

Section: Resultssupporting

confidence: 69%

“…One prominent model in the field has MutL(α) joining MutS(α) to form MutS(α)-MutL(α) sliding clamps that diffuse along the DNA to interact with the strand-discrimination signal (β-clamp/PCNA or MutH) (16). Other models include trapping of MutS(α) clamps near the mismatch by MutL(α) followed by DNA looping or, alternately, MutS(α)-induced polymerization of MutL(α) along the DNA to reach the strand-discrimination signal (6,7,15,18,22). Some degree of localization to the mismatch is suggested by in vitro studies of eukaryotic MMR proteins, indicating that although MutLα can introduce nicks across long stretches of DNA, they occur preferentially in the vicinity of the mismatch (9,11,12).…”

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confidence: 99%

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MutL traps MutS at a DNA mismatch

Qiu

Sakato

Sacho

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (β-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins. Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatch DNA to allow the subsequent activation of MutL's endonuclease activity by β-clamp/PCNA remains elusive. In both prokaryotes and eukaryotes, MutS homologs undergo conformational changes to a mobile clamp state that can move away from the mismatch. However, the function of this MutS mobile clamp is unknown. Furthermore, whether the interaction with MutL leads to a mobile MutS-MutL complex or a mismatch-localized complex is hotly debated. We used single molecule FRET to determine that Thermus aquaticus MutL traps MutS at a DNA mismatch after recognition but before its conversion to a sliding clamp. Rather than a clamp, a conformationally dynamic protein assembly typically containing more MutL than MutS is formed at the mismatch. This complex provides a local marker where interaction with β-clamp/PCNA could distinguish parent/daughter strand identity. Our finding that MutL fundamentally changes MutS actions following mismatch detection reframes current thinking on MMR signaling processes critical for genomic stability.DNA mismatch repair | MutS | MutL | FRET

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Section: Resultssupporting

confidence: 82%

Section: Resultssupporting

confidence: 69%

mentioning

confidence: 99%

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MutL traps MutS at a DNA mismatch

Qiu

Sakato

Sacho

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Our SAXS results provided no evidence that MutL binding involves stabilization of a bent form of the DNA substrate. Our data, which indicate that DNA bending only acts in initial mispair recognition, support models in which MutL or MutS/MutL complexes migrate along unbent DNA after MutS recognizes a mispair and binds ATP (21,22,48,49). The eukaryotic system is likely similar, given that mutant Msh2 and Msh6 proteins, whose only clear biochemical defect is an inability to form sliding clamps, cause in vivo MMR defects (50).…”

Section: Discussionsupporting

confidence: 68%

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

Hura

Tsai

Claridge

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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DNA metabolism and processing frequently require transient or metastable DNA conformations that are biologically important but challenging to characterize. We use gold nanocrystal labels combined with small angle X-ray scattering to develop, test, and apply a method to follow DNA conformations acting in the Escherichia coli mismatch repair (MMR) system in solution. We developed a neutral PEG linker that allowed gold-labeled DNAs to be flashcooled and stored without degradation in sample quality. The 1,000-fold increased gold nanocrystal scattering vs. DNA enabled investigations at much lower concentrations than otherwise possible to avoid concentration-dependent tetramerization of the MMR initiation enzyme MutS. We analyzed the correlation scattering functions for the nanocrystals to provide higher resolution interparticle distributions not convoluted by the intraparticle distribution. We determined that mispair-containing DNAs were bent more by MutS than complementary sequence DNA (csDNA), did not promote tetramer formation, and allowed MutS conversion to a sliding clamp conformation that eliminated the DNA bends. Addition of second protein responder MutL did not stabilize the MutS-bent forms of DNA. Thus, DNA distortion is only involved at the earliest mispair recognition steps of MMR: MutL does not trap bent DNA conformations, suggesting migrating MutL or MutS/MutL complexes as a conserved feature of MMR. The results promote a mechanism of mismatch DNA bending followed by straightening in initial MutS and MutL responses in MMR. We demonstrate that small angle X-ray scattering with gold labels is an enabling method to examine protein-induced DNA distortions key to the DNA repair, replication, transcription, and packaging. D NA is frequently considered a passive component in interactions with proteins involved in DNA metabolism. Despite this view, many proteins use DNA structural features to mediate catalysis and identify damaged DNA through the effects of damage on DNA rigidity and conformation (1-6). The view of DNA as a passive element is therefore at least in part due to a paucity of robust tools to examine dynamic DNA conformational states during multistep reactions. Gold-labeled DNA enables measurement over length scales sufficient to accommodate several proteins to identify cooperative effects on DNA. Because X-rays scatter predominantly from electrons, using heavy atom labels (7-11) provides high contrast relative to organic molecules. By using labels of moderate size (∼5 nm), the scattering from gold nanocrystals dominates all other scattering signals by three orders of magnitude, thereby reducing analysis complexity while minimizing nanocrystal influence on biological macromolecules. Importantly, small angle X-ray scattering (SAXS) provides global information on conformations adopted by a population of macromolecules in almost any solution condition (12)(13)(14).Mismatch repair (MMR) is an evolutionarily conserved process that corrects mismatches generated during DNA replication (15,16). Despite the i...

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“…A similar trend (i.e., mutL Ϸ mutS Ϸ mutSL Ͼ WT) in mutation frequency was observed for MMR mutants constructed in HG003 (Table 3). The increases in mutation frequency displayed by S. aureus MMR strain 8325 and HG003 mutants were modest and more than a log lower than those seen with similar mutations constructed in E. coli (27). This difference may be a result of increased DNA replication fidelity in S. aureus, although no reports currently exist showing higher fidelity with Gram-positive replicative polymerase PolIIIC.…”

Section: Resultsmentioning

confidence: 79%

Evolution in Fast Forward: a Potential Role for Mutators in Accelerating Staphylococcus aureus Pathoadaptation

Canfield¹,

Schwingel²,

Foley³

et al. 2012

Journal of Bacteriology

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Pathogen evolution and subsequent phenotypic heterogeneity during chronic infection are proposed to enhance Staphylococcus aureus survival during human infection. We tested this theory by genetically and phenotypically characterizing strains with mutations constructed in the mismatch repair (MMR) and oxidized guanine (GO) system, termed mutators, which exhibit increased spontaneous-mutation frequencies. Analysis of these mutators revealed not only strain-dependent increases in the spontaneous-mutation frequency but also shifts in mutational type and hot spots consistent with loss of GO or MMR functions. Although the GO and MMR systems are relied upon in some bacterial species to prevent reactive oxygen species-induced DNA damage, no deficit in hydrogen peroxide sensitivity was found when either of these DNA repair pathways was lost in S. aureus. To gain insight into the contribution of increased mutation supply to S. aureus pathoadaptation, we measured the rate of ␣-hemolysin and staphyloxanthin inactivation during serial passage. Detection of increased rates of ␣-hemolysin and staphyloxanthin inactivation in GO and MMR mutants suggests that these strains are capable of modifying virulence phenotypes implicated in mediating infection. Accelerated derivation of altered virulence phenotypes, combined with the absence of increased ROS sensitivity, highlights the potential of mutators to drive pathoadaptation in the host and serve as catalysts for persistent infections.

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Stoichiometry of MutS and MutL at unrepaired mismatches in vivo suggests a mechanism of repair

Cited by 43 publications

References 39 publications

MutL traps MutS at a DNA mismatch

MutL traps MutS at a DNA mismatch

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

Evolution in Fast Forward: a Potential Role for Mutators in Accelerating Staphylococcus aureus Pathoadaptation

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