ISOLATION OF MUTS-BETA FROM HUMAN-CELLS AND COMPARISON OF THE MISMATCH REPAIR SPECIFICITIES OF MUTS-BETA AND MUTS-ALPHA

A human MSH2-human MSH3 (hMSH2 . hMSH3) complex of approximately 1:1 s toichiometry (human MutS beta (hMutS beta)) has been demonstrated in s everal human tumor cell lines and purified to near homogeneity, In vit ro, hMutS beta supports the efficient repair of insertion/deletion (I/ D) heterologies of 2-8 nucleotides, is weakly active on a single-nucle otide I/D mispair, and is not detectably active on the eight base-base mismatches, Human MutS alpha (hMutS alpha), a heterodimer of hMSH2 an d hMSH6, efficiently supports the repair of single-nucleotide IID mism atches, base-base mispairs, and all substrates tested that were repair ed by hMutS beta, Thus, the repair specificities of hMutS alpha and hM utS beta are redundant with respect to the repair of I/D heterologies of 2-8 nucleotides. The hMutSa level in repair-proficient HeLa cells ( 1.5 mu g/mg nuclear extract) is approximately 10 times that of hMutS b eta. In HCT-15 colorectal tumor cells, which do not contain hMSH6 and consequently lack hMutS alpha, the hMutS beta level is elevated severa lfold relative to that in HeLa cells and is responsible for the repair of I/D mismatches that has been observed in this cell line. LoVo tumo r cells, which are genetically deficient in hMSH2, lack both hMutS alp ha and hMutS beta, and hMSH3 and hMSH6 levels are less than 4% of thos e found in repair-proficient cells. Coupled with previous findings (J. T. Drummond, J, Genschel, E. Wolf, and P. Modrich (1997) Proc. Natl. Acad Sci. U.S.A. 94, 10144-10149), these results suggest that hMSH2 pa rtitions between available pools of hMSH3 and hMSH6 and indicate that hMSH2 positively modulates hMSH6 and hMSH3 levels, perhaps by stabiliz ation of the polypeptides upon heterodimer formation.