scholarly journals A Lynch syndrome-associated mutation at a Bergerat ATP-binding fold destabilizes the structure of the DNA mismatch repair endonuclease MutL

2020 ◽  
Vol 295 (33) ◽  
pp. 11643-11655
Author(s):  
Keisuke Izuhara ◽  
Kenji Fukui ◽  
Takeshi Murakawa ◽  
Seiki Baba ◽  
Takashi Kumasaka ◽  
...  
Keyword(s):  
Dna Binding ◽  
Lynch Syndrome ◽  
Atpase Activity ◽  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
Limited Proteolysis ◽  
Atp Binding ◽  
Aquifex Aeolicus ◽  
Dna Mismatch ◽  
Genes Encoding

In humans, mutations in genes encoding homologs of the DNA mismatch repair endonuclease MutL cause a hereditary cancer that is known as Lynch syndrome. Here, we determined the crystal structures of the N-terminal domain (NTD) of MutL from the thermophilic eubacterium Aquifex aeolicus (aqMutL) complexed with ATP analogs at 1.69–1.73 Å. The structures revealed significant structural similarities to those of a human MutL homolog, postmeiotic segregation increased 2 (PMS2). We introduced five Lynch syndrome-associated mutations clinically found in human PMS2 into the aqMutL NTD and investigated the protein stability, ATPase activity, and DNA-binding ability of these protein variants. Among the mutations studied, the most unexpected results were obtained for the residue Ser34. Ser34 (Ser46 in PMS2) is located at a previously identified Bergerat ATP-binding fold. We found that the S34I aqMutL NTD retains ATPase and DNA-binding activities. Interestingly, CD spectrometry and trypsin-limited proteolysis indicated the disruption of a secondary structure element of the S34I NTD, destabilizing the overall structure of the aqMutL NTD. In agreement with this, the recombinant human PMS2 S46I NTD was easily digested in the host Escherichia coli cells. Moreover, other mutations resulted in reduced DNA-binding or ATPase activity. In summary, using the thermostable aqMutL protein as a model molecule, we have experimentally determined the effects of the mutations on MutL endonuclease; we discuss the pathological effects of the corresponding mutations in human PMS2.

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.

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

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.

Prevalence and molecular characteristics of DNA mismatch repair deficient endometrial cancer in a Japanese hospital-based population

2020 ◽  
Vol 51 (1) ◽  
pp. 60-69 ◽  
Author(s):  
Azusa Yamamoto ◽  
Tatsuro Yamaguchi ◽  
Okihide Suzuki ◽  
Tetsuya Ito ◽  
Noriyasu Chika ◽  
...  
Keyword(s):  
Endometrial Cancer ◽  
Lynch Syndrome ◽  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
Molecular Characteristics ◽  
Variant Of Uncertain Significance ◽  
Dna Mismatch ◽  
Germline Variant ◽  
Uncertain Significance ◽  
Repair Genes

Abstract Background The prevalence and molecular characteristics of defective DNA mismatch repair endometrial cancers in the Japanese population have been underexplored. Data supporting clinical management of patients with Lynch-like syndrome and germline variant of uncertain significance of mismatch repair genes are still lacking. Methods Immunohistochemistry of mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) was performed on formalin-fixed paraffin-embedded sections prepared from resected primary endometrial cancers in 395 women with a median age of 59 years. Genetic and/or epigenetic alterations of the mismatch repair genes were also investigated. Results Loss of expression of one or more mismatch repair proteins was observed in 68 patients (17.2%). A total of 17 out of 68 patients (25%, 4.3% of all cases) were identified as candidates for genetic testing for Lynch syndrome after excluding 51 patients with MLH1 hypermethylated cancer. Fourteen of these 17 patients subjected to genetic testing were found to have Lynch syndrome (n = 5), germline variant of uncertain significance (n = 2) or Lynch-like syndrome (n = 7). Compared with patients with Lynch syndrome, those with germline variant of uncertain significance and Lynch-like syndrome tended to demonstrate an older age at the time of endometrial cancer diagnosis (P = 0.07), less fulfillment of the revised Bethesda guidelines (P = 0.09) and lower prevalence of Lynch syndrome-associated tumors in their first-degree relatives (P = 0.01). Conclusions This study provides useful information for management in patients with DNA mismatch repair endometrial cancer. Specifically, cancer surveillance as recommended in patients with Lynch syndrome might not be necessary in patients with germline variant of uncertain significance and Lynch-like syndrome and their relatives.

DNA mismatch repair and Lynch syndrome

2006 ◽  
Vol 37 (5-7) ◽  
pp. 271-283 ◽  
Author(s):  
Guido Plotz ◽  
Stefan Zeuzem ◽  
Jochen Raedle
Keyword(s):  
Lynch Syndrome ◽  
Mismatch Repair ◽  
Dna Mismatch Repair ◽  
Dna Mismatch
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