Multiple mutations and frameshifts are the hallmark of defective hPMS2 in pZ189-transfected human tumor cells

Two HeLa variants defective in the mismatch repair protein hPMS2 were isola ted by selection for methylation tolerance. Neither variant expressed detec table hPMS2 protein as determined by western blotting. Cell extracts were d efective in correcting a single base mispair and were unable to perform mis match repair-dependent processing of a methylated DNA substrate. Correction of the repair defect and restoration of sensitivity to a methylating agent was achieved by introducing a wild-type copy of chromosome 7 on which the hPMS2 gene is located. Loss of hPMS2 function in the HeLa variants was asso ciated with a 5-fold increase in mutation frequency in the supF gene of the pZ189 shuttle vector. Wild-type levels of mutagenesis were restored by the transferred chromosome 7, Comparisons of mutational spectra identified mul tiple base substitutions, frameshifts and, to a lesser extent, single base pair changes as the types of mutation which are selectively increased in a hPMS2-defective background, The location of multiple mutations and frameshi fts indicates that misalignment-mediated mutagenesis could underlie most of these events. Thus the mutator phenotype associated with loss of hPMS2 mos t likely arises because of the failure to correct replication slippage erro rs. Our data also suggest that a considerable fraction of mutagenic interme diates are recognized by the hMutS beta complex and processed via the hMLH1 /hPMS2 heterodimer.