Marc Wormek | PubMed Health

PubMed2005

Analysis of the human MutLalpha.MutSalpha complex.

Human DNA mismatch repair is initiated by MutSalpha which ATP-dependently recruits MutLalpha. Analysis of this complex is difficult due to its transient and dynamic nature. We have optimized conditions for investigation of MutLalpha.MutSalpha complexes using a DNA pulldown assay. Non-specific DNA end-binding, which frequently interfered with analysis of the interaction, did not occur under the applied conditions. MutSalpha had significantly higher affinity to DNA mispairs, but its interaction with MutLalpha did not require a mismatch. Complex formation was best supported by low magnesium concentration and low temperature at physiological pH and salt concentration. Complex formation was delayed by the slowly hydrolyzable ATP analog ATPgammaS, undetectable with the non-hydrolyzable analog AMP-PNP, and occurred weakly with a combination of AMP-PNP and ADP, confirming that hydrolysis was required. The described conditions likely capture an intermediate of the repair reaction which has bound ATP and ADP in the two nucleotide-binding sites of MutSalpha.

Adenosine Triphosphate↗

PubMed2005

BRAF mutations in colorectal carcinoma suggest two entities of microsatellite-unstable tumors.

BACKGROUND: Alterations of BRAF have been implicated in the carcinogenesis of colorectal tumors with microsatellite instability (MSI). These alterations were attributed to defective DNA mismatch repair, which underlies MSI. It was the objective of this study to clarify the role of BRAF in colorectal carcinoma with MSI. METHODS: After sequencing for BRAF and KRAS in 82 colorectal tumor samples with or without MSI, mismatch repair protein expression was analyzed by immunohistochemistry, and promoter methylation of hMLH1 was analyzed with a methylation-specific polymerase chain reaction. Results were correlated with the germline status of hMLH1 or hMSH2 and clinical characteristics. RESULTS: BRAF was mutated more often in tumors with MSI than in tumors without MSI (27% vs. 5%; P = 0.016). The most prevalent BRAF alteration, V599E, occurred only in tumors with MSI. BRAF V599E was associated with more frequent hMLH1 promoter methylation (P = 0.07) and loss of hMLH1 (P = 0.02). The median age of patients with BRAF V599E was older compared with the median age of patients without this mutation (P = 0.001; 78 vs. 49 yrs). No BRAF alterations occurred in patients with germline mutations of mismatch repair genes. Five novel BRAF mutations were identified. CONCLUSIONS: Although BRAF V599E was common in colorectal carcinomas with MSI, it was not a consequence of deficient mismatch repair. The current data showed instead that the BRAF V599E mutation was associated only with a subgroup of colorectal carcinomas with MSI that were obtained from older patients without hereditary nonpolyposis colorectal carcinoma and showed epigenetic silencing of hMLH1. These results indicated that tumors with MSI caused by epigenetic MLH1 silencing have a distinct mutational background from that of tumors with genetic loss of mismatch repair, suggesting that there are two genetically distinct entities of microsatellite-instable tumors.

Adaptor Proteins, Signal Transducing↗