scholarly journals Structural characterization of transient interactions in DNA mismatch repair

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.

scholarly journals Functional Studies on the Candidate ATPase Domains of Saccharomyces cerevisiae MutLα

2000 ◽  
Vol 20 (17) ◽  
pp. 6390-6398 ◽  
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.

In vivo DNA mismatch repair measurement in zebrafish embryos and its use in screening of environmental carcinogens

2016 ◽  
Vol 302 ◽  
pp. 296-303 ◽  
Author(s):  
Yuanhong Chen ◽  
Changjiang Huang ◽  
Chenglian Bai ◽  
Changchun Du ◽  
Junhua Liao ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
Zebrafish Embryos ◽  
Environmental Carcinogens ◽  
Dna Mismatch

Cadmium inhibits human DNA mismatch repair in vivo

2004 ◽  
Vol 321 (1) ◽  
pp. 21-25 ◽  
Author(s):  
Anne Lützen ◽  
Sascha Emilie Liberti ◽  
Lene Juel Rasmussen
Keyword(s):  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
Dna Mismatch ◽  
Human Dna

scholarly journals DNA Mismatch Repair in Eukaryotes and Bacteria

2010 ◽  
Vol 2010 ◽  
pp. 1-16 ◽  
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.

scholarly journals Gene Expression Analysis of Tumor Spheroids Reveals a Role for Suppressed DNA Mismatch Repair in Multicellular Resistance to Alkylating Agents

2004 ◽  
Vol 24 (15) ◽  
pp. 6837-6849 ◽  
Author(s):  
Giulio Francia ◽  
Shan Man ◽  
Beverly Teicher ◽  
Luigi Grasso ◽  
Robert S. Kerbel
Keyword(s):  
Drug Resistance ◽  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
Alkylating Agents ◽  
Acquired Resistance ◽  
Dominant Negative ◽  
Tumor Spheroids ◽  
Dna Mismatch ◽  
Multicellular Resistance

ABSTRACT Drug resistance is a major obstacle in the successful treatment of cancer. Thus, elucidation of the mechanisms responsible is a critical first step in trying to prevent or delay such manifestations of resistance. In this regard, three-dimensional multicellular tumor cell spheroids are intrinsically more resistant to virtually all anticancer cytotoxic drugs than conventional monolayer cultures. We have employed the EMT-6 subline PC5T, which forms highly compact spheroids, and differential display to identify candidate genes whose expression differs between monolayer and spheroids. Approximately 5,000 bands were analyzed, revealing 26 to be differentially expressed. Analysis of EMT-6 tumor variants selected in vivo for acquired resistance to alkylating agents identified eight genes whose expression correlated with drug resistance in tumor spheroids. Four genes (encoding Nop56, the NADH SDAP subunit, and two novel sequences) were found to be down-regulated in EMT-6 spheroids and four (encoding 2-oxoglutarate carrier protein, JTV-1, and two novel sequences) were up-regulated. Analysis of the DNA mismatch repair-associated PMS2 gene, which overlaps at the genomic level with the JTV-1 gene, revealed PMS2 mRNA to be down-regulated in tumor spheroids, which was confirmed at the protein level. Analysis of PMS2−/− mouse embryo fibroblasts confirmed a role for PMS2 in sensitivity to cisplatin, and DNA mismatch repair activity was found to be reduced in EMT-6 spheroids compared to monolayers. Dominant negative PMS2 transfection caused increased resistance to cisplatin in EMT-6 and CHO cells. Our results implicate reduced DNA mismatch repair as a determinant factor of reversible multicellular resistance of tumor cells to alkylating agents.

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

2017 ◽  
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.

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

2020 ◽  
Author(s):  
Hu Fang ◽  
Xiaoqiang Zhu ◽  
Jieun Oh ◽  
Jayne A. Barbour ◽  
Jason W. H. Wong
Keyword(s):  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
Dna Polymerases ◽  
Induced Mutations ◽  
Mutational Signatures ◽  
Dna Mismatch ◽  
Mmr Deficiency ◽  
Mutational Process

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.

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

2005 ◽  
Vol 33 (4) ◽  
pp. 689-693 ◽  
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.

scholarly journals DNA Mismatch Repair Catalyzed by Extracts of Mitotic, Postmitotic, and Senescent Drosophila Tissues and Involvement of mei-9 Gene Function for Full Activity

1998 ◽  
Vol 18 (3) ◽  
pp. 1436-1443 ◽  
Author(s):  
Arvinder Bhui-Kaur ◽  
Myron F. Goodman ◽  
John Tower
Keyword(s):  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
Specific Activity ◽  
Excision Repair ◽  
Crossing Over ◽  
Dna Mismatch ◽  
Nucleotide Excision ◽  
Repair Activity ◽  
Development And Aging

ABSTRACT Extracts of Drosophila embryos and adults have been found to catalyze highly efficient DNA mismatch repair, as well as repair of 1- and 5-bp loops. For mispairs T · G and G · G, repair is nick dependent and is specific for the nicked strand of heteroduplex DNA. In contrast, repair of A · A, C · A, G · A, C · T, T · T, and C · C is not nick dependent, suggesting the presence of glycosylase activities. For nick-dependent repair, the specific activity of embryo extracts was similar to that of extracts derived from the entirely postmitotic cells of young and senescent adults. Thus, DNA mismatch repair activity is expressed in Drosophila cells during both development and aging, suggesting that there may be a function or requirement for mismatch repair throughout the Drosophila life span. Nick-dependent repair was reduced in extracts of animals mutant for themei-9 gene. mei-9 has been shown to be required in vivo for certain types of DNA mismatch repair, nucleotide excision repair (NER), and meiotic crossing over and is theDrosophila homolog of the yeast NER gene rad1.

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