title: DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSalpha/hMutSbeta ratio and reduces the efficiency of base-base mismatch repair.
abstract: 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 hMutSalpha (hMSH2. hMSH6) and hMutSbeta (hMSH2.hMSH3). In HL-60 extracts the hMutSalpha to hMutSbeta 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 hMutSalpha and hMutSbeta display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion-deletion heterology, but only hMutSalpha restores base-base mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.
authors.affiliation: Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: J T
authors.initials: JT
authors.lastname: Drummond
authors.forename: J
authors.initials: J
authors.lastname: Genschel
authors.forename: E
authors.initials: E
authors.lastname: Wolf
authors.forename: P
authors.initials: P
authors.lastname: Modrich
mesh_terms: D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins; D004352:Drug Resistance, Microbial / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D005784:Gene Amplification; D018922:HL-60 Cells; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009692:Nucleic Acid Heteroduplexes; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate