MISMATCH REPAIR DEFICIENCY ASSOCIATED WITH OVEREXPRESSION OF THE MSH3GENE

We tested the ability of recombinant hMutS alpha (hMSH2/hMSH6) and hMu tS beta (hMSH2/hMSH3) heterodimers to complement the mismatch repair d efect of HEC59, a human cancer cell line whose extracts lack all three MutS homologues. Although repair of both base/base mispairs and inser tion-deletion loops was restored by hMutS alpha, only the latter subst rates were addressed in extracts supplemented with hMutS beta. hMutS a lpha was also able to complement a defect in the repair of base/base m ispairs in CHO R and HL60R cell extracts. In these cells, methotrexate -induced amplification of the dihydrofolate reductase (DHFR) locus, wh ich also contains the MSH3 gene, led to an overexpression of MSH3 and thus to a dramatic change in the relative levels of MutS alpha and Mut S beta. As a rule, MSH2 is primarily complexed with MSH6. MutS alpha i s thus relatively abundant in mammalian cell extracts, whereas MutS be ta levels are generally low. In contrast, in cells that overexpress MS H3, the available MSH2 protein is sequestered predominantly into MutS beta. This leads to degradation of the partnerless MSH6 and depletion of MutS alpha. CHO R and HL60R cells therefore lack correction of base /base mispairs, whereas loop repair is maintained by MutS beta. Conseq uently, frameshift mutations in CHO R are rare, whereas transitions an d transversions are acquired at a rate two orders of magnitude above b ackground. Our data thus support and extend the findings of Drummond c t al. [Drummond, J. T., Genschel, J., Wolf, E. & Modrich, P. (1997) Pr oc. Natl. Acad. Sci. USA 94, 10144-10149] and demonstrate that mismatc h repair deficiency can arise not only through mutation or transcripti onal silencing of a mismatch repair gene, but also as a result of imba lance in the relative amounts of the MSH3 and MSH6 proteins.