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N-terminus of hMLH1 confers interaction of hMutL{alpha} and hMutL{beta} with hMutS{alpha}
(2003)
- Mismatch repair is a highly conserved system that ensures replication fidelity by repairing mispairs after DNA synthesis. In humans, the two protein heterodimers hMutS{alpha} (hMSH2-hMSH6) and hMutL{alpha} (hMLH1-hPMS2) constitute the centre of the repair reaction. After recognising a DNA replication error, hMutS{alpha} recruits hMutL{alpha}, which then is thought to transduce the repair signal to the excision machinery. We have expressed an ATPase mutant of hMutL{alpha} as well as its individual subunits hMLH1 and hPMS2 and fragments of hMLH1, followed by examination of their interaction properties with hMutS{alpha} using a novel interaction assay. We show that, although the interaction requires ATP, hMutL{alpha} does not need to hydrolyse this nucleotide to join hMutS{alpha} on DNA, suggesting that ATP hydrolysis by hMutL{alpha} happens downstream of complex formation. The analysis of the individual subunits of hMutL{alpha} demonstrated that the hMutS{alpha}–hMutL{alpha} interaction is predominantly conferred by hMLH1. Further experiments revealed that only the N-terminus of hMLH1 confers this interaction. In contrast, only the C-terminus stabilised and co-immunoprecipitated hPMS2 when both proteins were co-expressed in 293T cells, indicating that dimerisation and stabilisation are mediated by the C-terminal part of hMLH1. We also examined another human homologue of bacterial MutL, hMutL{beta} (hMLH1–hPMS1). We show that hMutL{beta} interacts as efficiently with hMutS{alpha} as hMutL{alpha}, and that it predominantly binds to hMutS{alpha} via hMLH1 as well.
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C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins
(2012)
- The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.
