Abstract
The DNA mismatch repair (MMR) pathway is essential in maintaining genomic stability through its role in DNA repair and the checkpoint response. Loss of DNA MMR underlies the hereditary cancer disease Lynch Syndrome (LS). Germline mutations in MSH2 account for approximately 40% of LS patients and of these, 18% are missense variants. One important clinical challenge has been discriminating between missense variants that are pathogenic and those that are not. Current analysis of missense mutations in MSH2 is performed using a combination of clinical, biochemical, and functional data; however, suitable cell culture models to test the various functions of the DNA MMR proteins are lacking. Here, we have generated human cell lines stably expressing a subset of MSH2 missense mutants and tested their effect on DNA repair and checkpoint response functions. We have expanded on previous biochemical and functional analyses performed in non-human systems to further understand defects conferred by this subset of single amino acid alterations. The functional characterization of MSH2 missense mutants combined with clinical and biochemical data is essential for appropriate patient management and genetic counseling decisions. ©2010 Wiley-Liss, Inc.
Highlights
The repair of single DNA nucleotide mismatches by the DNA mismatch repair (MMR) pathway is essential in maintaining genomic stability (Hsieh and Yamane, 2008; Li, 2008)
We have previously shown that MSH2 and MLH1 rapidly accumulate and form a protein complex with PCNA on the chromatin following treatment with MNNG (Mastrocola and Heinen, 2010)
To date the functional characterization of MSH2 missense mutations has been performed in model organisms such as bacteria and yeast or in vitro (Ellison, et al, 2001; Gammie, et al, 2007; Heinen, et al, 2002; Lützen, et al, 2008; Martinez and Kolodner, 2010; Ollila, et al, 2008; Ollila, et al, 2006)
Summary
The repair of single DNA nucleotide mismatches by the DNA mismatch repair (MMR) pathway is essential in maintaining genomic stability (Hsieh and Yamane, 2008; Li, 2008). Models of MMR mechanism propose that MSH2-MSH6 heterodimers load onto DNA via a mismatch and recruit downstream MMR factors including a heterodimer of the MutL homologues MLH1 and PMS2 (Acharya, et al, 2003; Constantin, et al, 2005; Zhang, et al, 2005). In addition to their role in the repair of replication errors, these MMR proteins are required for the Received 19 April 2010; accepted revised manuscript 15 July 2010. Activation of the DNA checkpoint response to low-doses of MNNG is mediated through the ATR-Chk pathway, which induces a G2 cell cycle arrest and subsequent apoptosis (Stojic, et al, 2004b)
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