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

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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The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

Nucleic Acids Research ◽

10.1093/nar/gkz834 ◽

2019 ◽

Vol 47 (22) ◽

pp. 11667-11680 ◽

Cited By ~ 5

Author(s):

Yannicka S N Mardenborough ◽

Katerina Nitsenko ◽

Charlie Laffeber ◽

Camille Duboc ◽

Enes Sahin ◽

...

Keyword(s):

Mismatch Repair ◽

Single Molecule ◽

Genome Stability ◽

Dna Mismatch Repair ◽

Dna Mismatch ◽

Replication Errors ◽

Dna Backbone ◽

Dna Strand ◽

Dna Replication Errors ◽

Gatc Site

Abstract DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here, we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.

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The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

10.1101/463133 ◽

2018 ◽

Cited By ~ 1

Author(s):

Yannicka SN Mardenborough ◽

Katerina Nitsenko ◽

Charlie Laffeber ◽

Camille Duboc ◽

Enes Sahin ◽

...

Keyword(s):

Mismatch Repair ◽

Single Molecule ◽

Genome Stability ◽

Dna Mismatch Repair ◽

Dna Mismatch ◽

Replication Errors ◽

Dna Backbone ◽

Dna Strand ◽

Dna Replication Errors ◽

Gatc Site

AbstractDNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.

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DNA Mismatch Repair in Eukaryotes and Bacteria

Journal of Nucleic Acids ◽

10.4061/2010/260512 ◽

2010 ◽

Vol 2010 ◽

pp. 1-16 ◽

Cited By ~ 83

Author(s):

Kenji Fukui

Keyword(s):

Mismatch Repair ◽

Molecular Mechanisms ◽

Dna Mismatch Repair ◽

Repair System ◽

Dna Mismatch ◽

Replication Errors ◽

Colon Cancers ◽

Basic Features ◽

Almost All ◽

Dna Replication Errors

DNA mismatch repair (MMR) corrects mismatched base pairs mainly caused by DNA replication errors. The fundamental mechanisms and proteins involved in the early reactions of MMR are highly conserved in almost all organisms ranging from bacteria to human. The significance of this repair system is also indicated by the fact that defects in MMR cause human hereditary nonpolyposis colon cancers as well as sporadic tumors. To date, 2 types of MMRs are known: the human type andEscherichia colitype. The basic features of the former system are expected to be universal among the vast majority of organisms including most bacteria. Here, I review the molecular mechanisms of eukaryotic and bacterial MMR, emphasizing on the similarities between them.

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Structural characterization of transient interactions in DNA mismatch repair

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314095813 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C418-C418

Author(s):

Monica Pillon ◽

Vignesh Babu ◽

Mark Sutton ◽

Alba Guarne

Keyword(s):

Complex Formation ◽

Mismatch Repair ◽

Structural Characterization ◽

Dna Mismatch Repair ◽

Full Length ◽

Endonuclease Activity ◽

Dimerization Domain ◽

Dna Mismatch ◽

Replication Errors

DNA mismatch repair (MMR) is a conserved pathway that safeguards genome integrity by correcting replication errors. The initiation of MMR is orchestrated by two proteins –MutS and MutL. MutS detects replication errors and recruits MutL, a key regulator in coordinating downstream MMR events. The processivity clamp, typically known to tether the replicative polymerase to DNA during DNA synthesis, also has a role in several steps in MMR. We have previously shown that MutL transiently interacts with the clamp and that this complex is important for MMR in vivo. The role of the clamp in eukaryotes and most bacteria is believed to license MutL endonuclease activity. In bacterial organisms where MutL does not have endonuclease activity, such as in Escherichia coli, the clamp also interacts with MutL and this interaction is also important for MMR activity. However, the transient nature of this complex prevents its functional and structural characterization. Here, we develop a method to stabilize the E. coli MutL-clamp complex by engineering a disulfide bond at the known protein complex interface and characterize its structure using small angle X-ray scattering (SAXS). MutL binds the clamp through a consensus motif found in its dimerization domain. Using this domain (MutL-CTD) we monitor complex formation with the clamp. We observe two complexes using SAXS. In one complex the MutL-CTD occupies a single hydrophobic cleft of the clamp, while the other occupies both hydrophobic clefts simultaneously. To identify the physiological complex, we used the full length MutL protein to impose further constraints. Analysis of complex formation suggests that full length MutL binds a single cleft on the clamp. Altogether, our data reveals how MutL interacts with the clamp in the early steps of MMR and this approach could be implemented to structurally characterize other transient complexes, an aspect of structural biology that is largely unexplored.

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Characterization of Pathogenic Human MSH2 Missense Mutations Using Yeast as a Model System: A Laboratory Course in Molecular Biology

Cell Biology Education ◽

10.1187/cbe.03-08-0006 ◽

2004 ◽

Vol 3 (1) ◽

pp. 31-48 ◽

Cited By ~ 10

Author(s):

Alison E. Gammie ◽

Naz Erdeniz

Keyword(s):

Molecular Biology ◽

Mismatch Repair ◽

Recombinant Dna ◽

Dna Mismatch Repair ◽

Genetic Material ◽

Molecular Techniques ◽

Original Research ◽

Missense Mutations ◽

Laboratory Course ◽

Dna Mismatch

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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Maternal Effect for DNA Mismatch Repair in the Mouse

Genetics ◽

10.1093/genetics/160.1.271 ◽

2002 ◽

Vol 160 (1) ◽

pp. 271-277

Author(s):

Vanessa E Gurtu ◽

Shelly Verma ◽

Allie H Grossmann ◽

R Michael Liskay ◽

William C Skarnes ◽

...

Keyword(s):

Mismatch Repair ◽

Maternal Effect ◽

Genome Stability ◽

Germ Line ◽

Dna Mismatch Repair ◽

Surprising Result ◽

Wild Type ◽

Repair Gene ◽

Dna Mismatch ◽

Dna Repair Gene

Abstract DNA mismatch repair (DMR) functions to maintain genome stability. Prokaryotic and eukaryotic cells deficient in DMR show a microsatellite instability (MSI) phenotype characterized by repeat length alterations at microsatellite sequences. Mice deficient in Pms2, a mammalian homolog of bacterial mutL, develop cancer and display MSI in all tissues examined, including the male germ line where a frequency of ~10% was observed. To determine the consequences of maternal DMR deficiency on genetic stability, we analyzed F1 progeny from Pms2−/− female mice mated with wild-type males. Our analysis indicates that MSI in the female germ line was ~9%. MSI was also observed in paternal alleles, a surprising result since the alleles were obtained from wild-type males and the embryos were therefore DMR proficient. We propose that mosaicism for paternal alleles is a maternal effect that results from Pms2 deficiency during the early cleavage divisions. The absence of DMR in one-cell embryos leads to the formation of unrepaired replication errors in early cell divisions of the zygote. The occurrence of postzygotic mutation in the early mouse embryo suggests that Pms2 deficiency is a maternal effect, one of a limited number identified in the mouse and the first to involve a DNA repair gene.

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Mutational signatures of DNA mismatch repair deficiency in C. elegans and human cancers

10.1101/149153 ◽

2017 ◽

Cited By ~ 2

Author(s):

B Meier ◽

NV Volkova ◽

Y Hong ◽

P Schofield ◽

PJ Campbell ◽

...

Keyword(s):

Mismatch Repair ◽

Dna Mismatch Repair ◽

Mutational Signatures ◽

Dna Mismatch ◽

C Elegans ◽

Replication Errors ◽

Human Cancers ◽

Repair Deficiency ◽

Mmr Deficiency ◽

Mutational Processes

ABSTRACTThroughout their lifetime cells are subject to extrinsic and intrinsic mutational processes leaving behind characteristic signatures in the genome. DNA mismatch repair (MMR) deficiency leads to hypermutation and is found in different cancer types. While it is possible to associate mutational signatures extracted from human cancers with possible mutational processes the exact causation is often unknown. Here we use C. elegans genome sequencing of pms-2 and mlh-1 knockouts to reveal the mutational patterns linked to C. elegans MMR deficiency and their dependency on endogenous replication errors and errors caused by deletion of the polymerase ε subunit pole-4. Signature extraction from 215 human colorectal and 289 gastric adenocarcinomas revealed three MMR-associated signatures, one of which closely resembles the C. elegans MMR spectrum and strongly discriminates microsatellite stable and unstable tumors (AUC=98%). A characteristic difference between human and C. elegans MMR deficiency is the lack of elevated levels of NCG>NTG mutations in C. elegans, likely caused by the absence of cytosine (CpG) methylation in worms. The other two human MMR signatures may reflect the interaction between MMR deficiency and other mutagenic processes, but their exact cause remains unknown. In summary, combining information from genetically defined models and cancer samples allows for better aligning mutational signatures to causal mutagenic processes.

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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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