Mutation spectrum of MSH3-deficient HHUA/chr.2 cells reflects in vivo activity of the MSH3 gene product in mismatch repair

The endometrial tumor cell line HHUA carries mutations in two mismatch repa ir (MMR) genes MSH3 and MSH6. We have established an MSH3-deficient HHUA/ch r.2 cell line by introducing human chromosome 2, which carries wild-type MS H6 and MSH2 genes, to HHUA cells. Introduction of chromosome 2 to HHUA cell s partially restored G:G MMR activity to the cell extract and reduced the f requency of mutation at the hypoxanthine-guanine phosphoribosyltransferase (hprt*) locus to about 3% that of the parental HHUA cells, which is five-fo ld the frequency in MMR-proficient cells, indicating that the residual muta tor activity in HHUA/chr.2 is due to an MSH3-deficiency in these cells. The spectrum of mutations occurring at the HPRT locus of HHUA/chr.2 was determ ined with 71 spontaneous 6TG(r) clones. Base substitutions and +/-1 bp fram eshifts were the major mutational events constituting, respectively, 54% an d 42% of the total mutations, and more than 70% of them occurred at A:T sit es. A possible explanation for the apparent bias of mutations to A:T sites in HHUA/chr.2 is haploinsufficiency of the MSH6 gene on the transferred chr omosome 2. Comparison of the mutation spectra of HHUA/chr.2 with that of th e MSH6-deficient HCT-15 cell Line [S. Ohzeki, A. Tachibana, K. Tatsumi, T. Kato, Carcinogenesis 18 (1997) 1127-1133.] suggests that in vivo the MutS a lpha (MSH2:MSH6) efficiently repairs both mismatch and unpaired extrahelica l bases, whereas MutS beta (MSH2:MSH3) efficiently repairs extrahelical bas es and repairs mismatch bases to a limited extent. (C) 2000 Elsevier Scienc e B.V. All rights reserved.