The mechanics of DNA mismatch repair sliding clamp progression in the prediction of clinically relevant HsMLH1 missense mutations

This work describes the project for an advanced undergraduate laboratory course in cell and molecular biology. One objective of the course is to teach students a variety of cellular and molecular techniques while conducting original research. A second objective is to provide instruction in science writing and data presentation by requiring comprehensive laboratory reports modeled on the primary literature. The project for the course focuses on a gene, MSH2, implicated in the most common form of inherited colorectal cancer. Msh2 is important for maintaining the fidelity of genetic material where it functions as an important component of the DNA mismatch repair machinery. The goal of the project has two parts. The first part is to create mapped missense mutation listed in the human databases in the cognate yeast MSH2 gene and to assay for defects in DNA mismatch repair. The second part of the course is directed towards understanding in what way are the variant proteins defective for mismatch repair. Protein levels are analyzed to determine if the missense alleles display decreased expression. Furthermore, the students establish whether the Msh2p variants are properly localized to the nucleus using indirect immunofluorescence and whether the altered proteins have lost their ability to interact with other subunits of the MMR complex by creating recombinant DNA molecules and employing the yeast 2-hybrid assay.

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The properties of Msh2–Msh6 ATP binding mutants suggest a signal amplification mechanism in DNA mismatch repair

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.005439 ◽

2018 ◽

Vol 293 (47) ◽

pp. 18055-18070 ◽

Cited By ~ 5

Author(s):

William J. Graham ◽

Christopher D. Putnam ◽

Richard D. Kolodner

Keyword(s):

Mismatch Repair ◽

Binding Sites ◽

Signal Amplification ◽

Dna Mismatch Repair ◽

Nucleotide Binding ◽

Atp Binding ◽

Dna Mismatch ◽

Nucleotide Binding Sites ◽

Sliding Clamp ◽

Atp Concentration

DNA mismatch repair (MMR) corrects mispaired DNA bases and small insertion/deletion loops generated by DNA replication errors. After binding a mispair, the eukaryotic mispair recognition complex Msh2–Msh6 binds ATP in both of its nucleotide-binding sites, which induces a conformational change resulting in the formation of an Msh2–Msh6 sliding clamp that releases from the mispair and slides freely along the DNA. However, the roles that Msh2–Msh6 sliding clamps play in MMR remain poorly understood. Here, using Saccharomyces cerevisiae, we created Msh2 and Msh6 Walker A nucleotide–binding site mutants that have defects in ATP binding in one or both nucleotide-binding sites of the Msh2–Msh6 heterodimer. We found that these mutations cause a complete MMR defect in vivo. The mutant Msh2–Msh6 complexes exhibited normal mispair recognition and were proficient at recruiting the MMR endonuclease Mlh1–Pms1 to mispaired DNA. At physiological (2.5 mm) ATP concentration, the mutant complexes displayed modest partial defects in supporting MMR in reconstituted Mlh1–Pms1-independent and Mlh1–Pms1-dependent MMR reactions in vitro and in activation of the Mlh1–Pms1 endonuclease and showed a more severe defect at low (0.1 mm) ATP concentration. In contrast, five of the mutants were completely defective and one was mostly defective for sliding clamp formation at high and low ATP concentrations. These findings suggest that mispair-dependent sliding clamp formation triggers binding of additional Msh2–Msh6 complexes and that further recruitment of additional downstream MMR proteins is required for signal amplification of mispair binding during MMR.

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Cryo-EM structures reveal how ATP and DNA binding in MutS coordinate the sequential steps of DNA mismatch repair

10.1101/2021.06.03.446775 ◽

2021 ◽

Author(s):

Alessandro Borsellini ◽

Vladislav Kunetsky ◽

Peter Friedhoff ◽

Meindert H. Lamers

Keyword(s):

Dna Binding ◽

Mismatch Repair ◽

Conformational Changes ◽

Atp Hydrolysis ◽

Dna Mismatch Repair ◽

Atp Binding ◽

Dna Mismatch ◽

Sliding Clamp ◽

Adp Release ◽

Atp Binding And Hydrolysis

DNA mismatch repair detects and removes mismatches from DNA reducing the error rate of DNA replication a 100-1000 fold. The MutS protein is one of the key players that scans for mismatches and coordinates the repair cascade. During this, MutS undergoes multiple conformational changes that initiate the subsequent steps, in response to ATP binding, hydrolysis, and release. How ATP induces the different conformations in MutS is not well understood. Here we present four cryo-EM structures of Escherichia coli MutS at sequential stages of the ATP hydrolysis cycle. These structures reveal how ATP binding and hydrolysis induces a closing and opening of the MutS dimer, respectively. Additional biophysical analysis furthermore explains how DNA binding modulates the ATPase cycle by preventing hydrolysis during scanning and mismatch binding, while preventing ADP release in the sliding clamp state. Nucleotide release is achieved when MutS encounters single stranded DNA that is produced during the removal of the daughter strand. This way, the combination of the ATP binding and hydrolysis and its modulation by DNA enable MutS to adopt different conformations needed to coordinate the sequential steps of the mismatch repair cascade.

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Functional consequences of DNA mismatch repair missense mutations in murine models and their impact on cancer predisposition

Biochemical Society Transactions ◽

10.1042/bst0330689 ◽

2005 ◽

Vol 33 (4) ◽

pp. 689-693 ◽

Cited By ~ 9

Author(s):

S.J. Scherer ◽

E. Avdievich ◽

W. Edelmann

Keyword(s):

Mismatch Repair ◽

Genome Stability ◽

Dna Mismatch Repair ◽

Significant Proportion ◽

Missense Mutations ◽

Dna Mismatch ◽

Replication Errors ◽

Tumour Suppression ◽

Functional Consequences ◽

Dna Replication Errors

Mutations in MMR (DNA mismatch repair) genes underlie HNPCC (hereditary non-polyposis colon cancer) and also a significant proportion of sporadic colorectal cancers. MMR maintains genome stability and suppresses tumour formation by correcting DNA replication errors and by mediating an apoptotic response to DNA damage. Analysis of mouse lines with MMR missense mutations demonstrates that these MMR functions can be separated and allows the assessment of their individual roles in tumour suppression. These studies in mice indicate that, although the increased mutation rates caused by MMR defects are sufficient to drive tumorigenesis, both functions co-operate in tumour suppression.

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Oligonucleotide-directed mutagenesis screen to identify pathogenic Lynch syndrome-associated MSH2 DNA mismatch repair gene variants

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1520813113 ◽

2016 ◽

Vol 113 (15) ◽

pp. 4128-4133 ◽

Cited By ~ 19

Author(s):

Hellen Houlleberghs ◽

Marleen Dekker ◽

Hildo Lantermans ◽

Roos Kleinendorst ◽

Hendrikus Jan Dubbink ◽

...

Keyword(s):

Lynch Syndrome ◽

Mismatch Repair ◽

Dna Mismatch Repair ◽

Embryonic Stem ◽

Mismatch Repair Gene ◽

Missense Mutations ◽

Repair Gene ◽

Dna Mismatch ◽

Mmr Genes ◽

Pathogenic Variants

Single-stranded DNA oligonucleotides can achieve targeted base-pair substitution with modest efficiency but high precision. We show that “oligo targeting” can be used effectively to study missense mutations in DNA mismatch repair (MMR) genes. Inherited inactivating mutations in DNA MMR genes are causative for the cancer predisposition Lynch syndrome (LS). Although overtly deleterious mutations in MMR genes can clearly be ascribed as the cause of LS, the functional implications of missense mutations are often unclear. We developed a genetic screen to determine the pathogenicity of these variants of uncertain significance (VUS), focusing on mutator S homolog 2 (MSH2). VUS were introduced into the endogenous Msh2 gene of mouse embryonic stem cells by oligo targeting. Subsequent selection for MMR-deficient cells using the guanine analog 6-thioguanine allowed the detection of MMR-abrogating VUS. The screen was able to distinguish weak and strong pathogenic variants from polymorphisms and was used to investigate 59 Msh2 VUS. Nineteen of the 59 VUS were identified as pathogenic. Functional assays revealed that 14 of the 19 detected variants fully abrogated MMR activity and that five of the detected variants attenuated MMR activity. Implementation of the screen in clinical practice allows proper counseling of mutation carriers and treatment of their tumors.

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

eLife ◽

10.7554/elife.06744 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 51

Author(s):

Flora S Groothuizen ◽

Ines Winkler ◽

Michele Cristóvão ◽

Alexander Fish ◽

Herrie HK Winterwerp ◽

...

Keyword(s):

Crystal Structure ◽

Dna Replication ◽

Mismatch Repair ◽

Conformational Changes ◽

Dna Mismatch Repair ◽

Transient State ◽

Dna Mismatch ◽

Sliding Clamp ◽

Downstream Effectors ◽

A Site

To avoid mutations in the genome, DNA replication is generally followed by DNA mismatch repair (MMR). MMR starts when a MutS homolog recognizes a mismatch and undergoes an ATP-dependent transformation to an elusive sliding clamp state. How this transient state promotes MutL homolog recruitment and activation of repair is unclear. Here we present a crystal structure of the MutS/MutL complex using a site-specifically crosslinked complex and examine how large conformational changes lead to activation of MutL. The structure captures MutS in the sliding clamp conformation, where tilting of the MutS subunits across each other pushes DNA into a new channel, and reorientation of the connector domain creates an interface for MutL with both MutS subunits. Our work explains how the sliding clamp promotes loading of MutL onto DNA, to activate downstream effectors. We thus elucidate a crucial mechanism that ensures that MMR is initiated only after detection of a DNA mismatch.

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