Deficiency in DNA mismatch repair of methylation damage is a major mutational process in cancer

AbstractDNA mismatch repair (MMR) is essential for maintaining genome integrity with its deficiency predisposing to cancer1. MMR is well known for its role in the post-replicative repair of mismatched base pairs that escape proofreading by DNA polymerases following cell division2. Yet, cancer genome sequencing has revealed that MMR deficient cancers not only have high mutation burden but also harbour multiple mutational signatures3, suggesting that MMR has pleotropic effects on DNA repair. The mechanisms underlying these mutational signatures have remained unclear despite studies using a range of in vitro4,5 and in vivo6 models of MMR deficiency. Here, using mutation data from cancer genomes, we identify a previously unknown function of MMR, showing that the loss of non-canonical replication-independent MMR activity is a major mutational process in human cancers. MMR is comprised of the MutSα (MSH2/MSH6) and MutLα (MLH1/PMS2) complexes7. Cancers with deficiency of MutSα exhibit mutational signature contributions distinct from those deficient of MutLα. This disparity is attributed to mutations arising from the unrepaired deamination of 5-methylcytosine (5mC), i.e. methylation damage, as opposed to replicative errors by DNA polymerases induced mismatches. Repair of methylation damage is strongly associated with H3K36me3 chromatin but independent of binding of MBD4, a DNA glycosylase that recognise 5mC and can repair methylation damage. As H3K36me3 recruits MutSα, our results suggest that MutSα is the essential factor in mediating the repair of methylation damage. Cell line models of MMR deficiency display little evidence of 5mC deamination-induced mutations as their rapid rate of proliferation limits for the opportunity for methylation damage. We thus uncover a non-canonical role of MMR in the protection against methylation damage in non-dividing cells.

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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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In vitro and in vivo modulations of benzo[c]phenanthrene-DNA adducts by DNA mismatch repair system

Nucleic Acids Research ◽

10.1093/nar/gkg875 ◽

2003 ◽

Vol 31 (22) ◽

pp. 6428-6434 ◽

Cited By ~ 12

Author(s):

J. Wu

Keyword(s):

Mismatch Repair ◽

Dna Adducts ◽

Dna Mismatch Repair ◽

Repair System ◽

Dna Mismatch ◽

Dna Mismatch Repair System ◽

Mismatch Repair System

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Isolation and Characterization of New Proliferating Cell Nuclear Antigen (POL30) Mutator Mutants That Are Defective in DNA Mismatch Repair

Molecular and Cellular Biology ◽

10.1128/mcb.22.19.6669-6680.2002 ◽

2002 ◽

Vol 22 (19) ◽

pp. 6669-6680 ◽

Cited By ~ 44

Author(s):

Patrick J. Lau ◽

Hernan Flores-Rozas ◽

Richard D. Kolodner

Keyword(s):

Amino Acid ◽

Mismatch Repair ◽

Proliferating Cell Nuclear Antigen ◽

Dna Mismatch Repair ◽

Nuclear Antigen ◽

Dna Mismatch ◽

Isolation And Characterization ◽

Proliferating Cell

ABSTRACT A number of studies have suggested a role for proliferating cell nuclear antigen (PCNA) in DNA mismatch repair (MMR). However, the majority of mutations in the POL30 gene encoding PCNA that cause MMR defects also cause replication and other repair defects that contribute to the increased mutation rate caused by these mutations. Here, 20 new pol30 mutants were identified and screened for MMR and other defects, resulting in the identification of two mutations, pol30-201 and pol30-204, that appear to cause MMR defects but little if any other defects. The pol30-204 mutation altered an amino acid (C81R) in the monomer-monomer interface region and resulted in a partial general MMR defect and a defect in MSH2-MSH6 binding in vitro. The pol30-201 mutation altered an amino acid (C22Y) located on the surface of the PCNA trimer that slides over the DNA but did not cause a defect in MSH2-MSH6 binding in vitro. The pol30-201 mutation caused an intermediate mutator phenotype. However, the pol30-201 mutation caused almost a complete defect in the repair of AC and GT mispairs and only a small defect in the repair of a “+T” insertion, an effect similar to that caused by an msh6Δ mutation, indicating that pol30-201 primarily effects MSH6-dependent MMR. The chromosomal double mutant msh3-FF>AA msh6-FF>AA eliminating the conserved FF residues of the PCNA interacting motif of these proteins caused a small (<10%) defect in MMR but showed synergistic interactions with mutations in POL30, indicating that the FF>AA substitution may not eliminate PCNA interactions in vivo. These results indicate that the interaction between PCNA and MMR proteins is more complex than was previously appreciated.

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Human DNA mismatch repair in vitro operates independently of methylation status at CpG sites

Nucleic Acids Research ◽

10.1093/nar/29.11.2234 ◽

2001 ◽

Vol 29 (11) ◽

pp. 2234-2243 ◽

Cited By ~ 12

Author(s):

J. T. Drummond

Keyword(s):

Mismatch Repair ◽

Dna Mismatch Repair ◽

Methylation Status ◽

Dna Mismatch ◽

Cpg Sites ◽

Human Dna

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Heterogeneity of DM kinase repeat expansion in different fetal tissues and further expansion during cell proliferation in vitro: evidence for a causal involvement of methyl-directed DNA mismatch repair in triplet repeat stability

Human Molecular Genetics ◽

10.1093/hmg/4.7.1147 ◽

1995 ◽

Vol 4 (7) ◽

pp. 1147-1153 ◽

Cited By ~ 60

Author(s):

D. Wohrle ◽

I. Kennerknecht ◽

M. Wolf ◽

H. Enders ◽

S. Schwemmle ◽

...

Keyword(s):

Cell Proliferation ◽

Mismatch Repair ◽

Dna Mismatch Repair ◽

Repeat Expansion ◽

Triplet Repeat ◽

Dna Mismatch ◽

Fetal Tissues ◽

Proliferation In Vitro

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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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Functional Studies on the Candidate ATPase Domains of Saccharomyces cerevisiae MutLα

Molecular and Cellular Biology ◽

10.1128/mcb.20.17.6390-6398.2000 ◽

2000 ◽

Vol 20 (17) ◽

pp. 6390-6398 ◽

Cited By ~ 59

Author(s):

Phuoc T. Tran ◽

R. Michael Liskay

Keyword(s):

Saccharomyces Cerevisiae ◽

Mismatch Repair ◽

Conformational Changes ◽

Atp Hydrolysis ◽

Dna Mismatch Repair ◽

Limited Proteolysis ◽

Atp Binding ◽

Dna Mismatch ◽

Functional Studies

ABSTRACT Saccharomyces cerevisiae MutL homologues Mlh1p and Pms1p form a heterodimer, termed MutLα, that is required for DNA mismatch repair after mismatch binding by MutS homologues. Recent sequence and structural studies have placed the NH2 termini of MutL homologues in a new family of ATPases. To address the functional significance of this putative ATPase activity in MutLα, we mutated conserved motifs for ATP hydrolysis and ATP binding in both Mlh1p and Pms1p and found that these changes disrupted DNA mismatch repair in vivo. Limited proteolysis with purified recombinant MutLα demonstrated that the NH2 terminus of MutLα undergoes conformational changes in the presence of ATP and nonhydrolyzable ATP analogs. Furthermore, two-hybrid analysis suggested that these ATP-binding-induced conformational changes promote an interaction between the NH2 termini of Mlh1p and Pms1p. Surprisingly, analysis of specific mutants suggested differential requirements for the ATPase motifs of Mlh1p and Pms1p during DNA mismatch repair. Taken together, these results suggest that MutLα undergoes ATP-dependent conformational changes that may serve to coordinate downstream events during yeast DNA mismatch repair.

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Trifluridine to enhance cytotoxicity by DNA mismatch repair deficiency and subsequent MBD4 frameshift mutation in colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2016.34.4_suppl.608 ◽

2016 ◽

Vol 34 (4_suppl) ◽

pp. 608-608 ◽

Cited By ~ 3

Author(s):

Satoshi Suzuki ◽

Moriya Iwaizumi ◽

Yasushi Hamaya ◽

Kosuke Takagaki ◽

Satoshi Osawa ◽

...

Keyword(s):

Colorectal Cancer ◽

Mismatch Repair ◽

Frameshift Mutation ◽

Dna Mismatch Repair ◽

Clonogenic Assay ◽

Dna Mismatch ◽

Cell Clones ◽

Human Colorectal Cancer Cell ◽

Mmr Deficiency ◽

Colorectal Cancer Patients

608 Background: TAS-102 is composed of trifluridine (FTD) and tipiracil hydrochloride and was shown to prolong survival of patients with refractory metastatic colorectal cancer (CRC). FTD is the active antitumor component of TAS102 and its metabolite TF-TTP chemically resembles 5FU-derived metabolite FdUTP in that both of them are misincorporated into DNA and lead to cytotoxicity. Several groups have reported that stage II-III colorectal cancer patients with tumors that lost DNA mismatch repair (MMR) function do not derive a benefit from 5-FU based chemotherapy. Although FTD is reported to be misincorporated into DNA, it is not known if MMR deficient CRC cells have chemoresistance for FTD. Methods: We first utilized human colorectal cancer cell lines HCT-116 (hMLH1-deficient cells) and HCT116+ch3 (hMLH1-retained cells), and compared cytotoxicity for FTD by clonogenic assay. To further analyze if 5FU refractory CRC cells have chemosensitivity for FTD, we established 5FU refractory HCT116 cells by continuous 5FU treatment for 10 month and analyzed cytotoxicity for FTD. Finally we constructed an expression plasmid of truncated DNA grycosylase MBD4, (MBD4tru) by frameshift mutation with MMR deficiency and stably transfected the construct into HCT116 CRC cells and selected HCT116MBD4tru cell clones. The HCT116MBD4tru cells were treated with FTD and analyzed for cytotoxicity by clonogenic assay. Results: In hMLH1-deficient cells, the number of colony was reduced by FTD treatment to a same degree of hMLH1-proficient cells whereas the number of colony by 5FU treatment is higher in hMLH1-deficitent cells than hMLH1-retained cells (p< 0.05). In 5FU refractory cells, treatment of FTD showed cytotoxicity to the same degree of non-5FU refractory cells. Interestingly, HCT116MBD4tru cells led to cytotoxicity with a higher sensitivity than control cells (p< 0.05). Conclusions: FTD induces cytotoxisity independently of MMR status as well as under 5FU refractory condition, and MBD4 frameshift mutation by MMR deficiency enhances FTD sensitivity. These results suggest that FTD may be useful for patients with MSI-H/MBD4 mutant CRC as well as for those with 5FU refractory CRC.

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