DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutS alpha/hMutS beta ratio and reduces the efficiency of base-base mismatch repair

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (DHFR) and MSH3 genes, Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutS alpha (hMSH2 . hMSH6) and hMutS beta (hMSH2 . hMSH3). In HL-60 extracts the hMutS alpha to hMutS beta ratio is roughly 6:1, whereas in methotrexate-resistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of base-base mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (HPRT) locus, Purified hMutS alpha and hMutS beta display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion-deletion heterology, but only hMutS alpha restores base-base mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.