MutS/MutL crystal structure reveals that the MutS sliding clamp loads MutL onto DNA

Groothuizen, F.S.; Winkler, Ines; Cristóvão, Michele; Fish, Alexander; Winterwerp, H.H.K.; Reumer, Annet; Marx, Andreas D.; Hermans, Nicolaas; Nicholls, Robert A.; Murshudov, Garib N.; Lebbink, Joyce H.G.; Friedhoff, Peter; Sixma, Titia K.

doi:10.7554/elife.06744

Cited by 96 publications

(171 citation statements)

References 72 publications

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“…A recently published study demonstrated that addition of E. coli MutL greatly reduces the rate at which MutS slides off mismatched DNA, consistent with our findings (41).…”

Section: Note Added In Proofsupporting

confidence: 93%

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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“…A recently published study demonstrated that addition of E. coli MutL greatly reduces the rate at which MutS slides off mismatched DNA, consistent with our findings (41).…”

Section: Note Added In Proofsupporting

confidence: 93%

MutL traps MutS at a DNA mismatch

Qiu

Sakato

Sacho

et al. 2015

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

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“…These functional data are in line with crystal structures of MutS proteins showing that the ATPase sites are located at the dimer interface and have a composite nature, with each subunit contributing residues to the site on the other subunit (Fig. 1) [50,51], and that ATP binding tightens the interface [26,43,52,53]. Bulk kinetic measurements of fluorophore-labeled Taq MutS domains I under the same reaction conditions reveal robust allosteric signaling between the ATPase and DNA binding sites despite their distant locations (~70 Å apart), with the protein flipping rapidly between two distinct conformations, one upon ATP binding to both subunits (10 6 M −1 s −1 at 40 °C) and the other upon ATP hydrolysis at the tight site (10 s −1 at 40 °C; Fig.…”

Section: Muts Actions In Mmrsupporting

confidence: 80%

“…3, 4c) [23,58]. In addition to domains I moving away from the mismatch [58], new structural data indicate that domains IV cross each other (keeping the clamp closed) and the connector domains II move outward [53]; hence, ATP-bound MutS can slide on DNA [41,59] without rotating along the helical contour, as confirmed recently by sm-tracking experiments with Taq and yeast MutS proteins [18-21,60]. It is surprising that after the effort of finding and marking a rare mismatch on DNA (akin to finding a needle in a haystack), MutS adopts a mobile state that can diffuse freely away from the MMR target site.…”

Section: Muts Actions In Mmrmentioning

confidence: 99%

Mismatch binding, ADP–ATP exchange and intramolecular signaling during mismatch repair

Hingorani

2016

DNA Repair

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The focus of this article is on the DNA binding and ATPase activities of the mismatch repair (MMR) protein, MutS—our current understanding of how this protein uses ATP to fuel its actions on DNA and initiate repair via interactions with MutL, the next protein in the pathway. Structure-function and kinetic studies have yielded detailed views of the MutS mechanism of action in MMR. How MutS and MutL work together after mismatch recognition to enable strand-specific nicking, which leads to strand excision and synthesis, is less clear and remains an active area of investigation.

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“…One of the key postulated benefits of sexual recombination is the reduction of Muller's ratchet. To determine whether the recombination-proficient strain can effectively achieve this during an ALE experiment, we generated a mutator version of our strain by deleting mutS, the mismatch recognition component of the mismatch repair system (48,49), and carried out an ALE experiment in rich medium (LB) for ϳ850 generations. The mutation rate of the ⌬mutS strain was increased by ϳ200-to 300-fold in both the genderless mutator and the asexual 2xoriT mutator strains over that of the nonmutator counterparts HFR-2xoriT-SFX and BW25113-2xoriT, respectively.…”

Section: Resultsmentioning

confidence: 99%

Benefits of a Recombination-Proficient Escherichia coli System for Adaptive Laboratory Evolution

Peabody

Winkler

Fountain

et al. 2016

Appl Environ Microbiol

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Adaptive laboratory evolution typically involves the propagation of organisms asexually to select for mutants with the desired phenotypes. However, asexual evolution is prone to competition among beneficial mutations (clonal interference) and the accumulation of hitchhiking and neutral mutations. The benefits of horizontal gene transfer toward overcoming these known disadvantages of asexual evolution were characterized in a strain of Escherichia coli engineered for superior sexual recombination (genderless). Specifically, we experimentally validated the capacity of the genderless strain to reduce the mutational load and recombine beneficial mutations. We also confirmed that inclusion of multiple origins of transfer influences both the frequency of genetic exchange throughout the chromosome and the linkage of donor DNA. We built a simple kinetic model to estimate recombination frequency as a function of transfer size and relative genotype enrichment in batch transfers; the model output correlated well with the experimental data. Our results provide strong support for the advantages of utilizing the genderless strain over its asexual counterpart during adaptive laboratory evolution for generating beneficial mutants with reduced mutational load. IMPORTANCEOver 80 years ago Fisher and Muller began a debate on the origins of sexual recombination. Although many aspects of sexual recombination have been examined at length, experimental evidence behind the behaviors of recombination in many systems and the means to harness it remain elusive. In this study, we sought to experimentally validate some advantages of recombination in typically asexual Escherichia coli and determine if a sexual strain of E. coli can become an effective tool for strain development.

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MutS/MutL crystal structure reveals that the MutS sliding clamp loads MutL onto DNA

Cited by 96 publications

References 72 publications

MutL traps MutS at a DNA mismatch

MutL traps MutS at a DNA mismatch

Mismatch binding, ADP–ATP exchange and intramolecular signaling during mismatch repair

Benefits of a Recombination-Proficient Escherichia coli System for Adaptive Laboratory Evolution

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