FUNCTIONAL GENETIC TESTS OF DNA MISMATCH REPAIR PROTEIN-ACTIVITY IN SACCHAROMYCES-CEREVISIAE

Hereditary non-polyposis colorectal cancer (HNPCC) is associated with mutations in four different genes encoding proteins involved in DNA mi smatch repair (DMR). As many as 30% of the observed sequence variation s in human DMR genes predict minor alterations in the encoded protein, such as amino acid (aa) replacements or small in-frame deletions/inse rtions. For such sequence variants, a functional genetic test will be required to discriminate mutations from polymorphisms. We have constru cted a series of isogenic yeast strains in which individual genes invo lved in DMR are disrupted, and have standardized an assay which measur es GT tract stability (Strand, M., Prolla, T.A., Liskay, R.M. and Fete s, T.D., 1993, Nature 365, 274-276) to characterize these gene product s. Disruptions of the yeast MSH2, MLH1, and PMS1 genes result in, resp ectively, a 290-, 450- and 390-fold increased tract instability over t he wild type (wt) strain under optimized assay conditions. Expression of the wt MSH2 and PMS1 gene from plasmids results in complementation of the corresponding chromosomal gene disruption. Two different aa rep lacements which correspond to previously observed sequence variants of the human MSH2 gene, and implicated in HNPCC, were created in the con served aa of the yeast MSH2 gene by site directed mutagenesis. Convers ion of the Pro(640) in the yeast protein to Leu resulted in a complete loss of protein function. In contrast, a yeast MSH2p protein in which the His(658) is changed to Tyr retains full function in this in vivo assay. These results indicate that the Pro --> Leu and His --> Tyr var iants observed in humans constitute, respectively, a mutation and a po lymorphism. The system described here may be used for further structur e/function analysis of yeast DMR proteins. Such studies may provide in sight into the effects that specific sequence variations observed in h uman DMR proteins have on their function. (C) 1998 Elsevier Science B. V. All rights reserved.