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PLoS By Category | Recent
PLoS Articles
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Biochemistry - Biophysics - Biotechnology - Oncology
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C-Terminal Fluorescent Labeling Impairs Functionality of DNA Mismatch Repair Proteins
Published:
Tuesday, February 14, 2012
Author:
Angela Brieger et al.
by Angela Brieger, Guido Plotz, Inga Hinrichsen, Sandra Passmann, Ronja Adam, Stefan Zeuzem
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 MutLa (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 MutLa's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLa. 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 MutLa. In contrast, N-terminal tagged MutLas retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.
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