Inefficient in vivo repair of mismatches at an oncogenic hotspot correlated with lack of binding by mismatch repair proteins and with phase of the cell cycle.
Repair rates of mismatched nucleotides located at an activating hotspot of mutation, H-ras codon 12, have been analyzed in vivo in mammalian cells. Repair rates at codon 12 are significantly improved in cells synchronized to the G(1) stage of the mammalian cell cycle as compared with non-synchronous cells, demonstrating that mismatch repair mechanisms are active in G(1). Repair rates in non-synchronous cells for the same mismatches at a nearby non-hotspot of mutation, H-ras codon 10, are also significantly improved over repair rates at codon 12 in non-synchronous cells, demonstrating that DNA mismatch repair rates can differ depending on the sequence context. These results suggest that inefficiencies in mismatch repair are responsible, at least in part, for the well documented hotspot of mutation at codon 12. Further experiments involving gel-shift analysis demonstrate a mismatch-specific binding factor for which the degree of binding correlates with in vivo repair rates for each mismatch tested at the codon 12 location. This binding factor appears to be the hMutSalpha heterodimer as identified by monoclonal antibody assay and inhibition of binding by ATP. Furthermore, a lack of binding is observed only for G:A mismatches at the codon 12 location. This lack of binding correlates with the low rate of repair observed in vivo for G:A mismatches at codon 12 versus the improved repair rates for G:A mismatches at codon 10. This may have biological relevance in that G:C-->T:A tranversions are a common mutation at this location in naturally occurring human tumors. These results suggest that there is lowered efficiency in the kinetics of mismatch repair at codon 12. Mismatches at this location are therefore more likely to be replicated before repair, thus resulting in a mutation.
Matton
N
N
Department of Biological Sciences/Biomedical Program, University of Alaska Anchorage, 3211 Providence Drive, Anchorage, AK 99508, USA.
Simonetti
J
J
Williams
K
K
D016475:3T3 Cells; D000818:Animals; D020137:Base Pair Mismatch / Q000235:genetics*; D003062:Codon / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D016193:G1 Phase / Q000235:genetics; D011905:Genes, ras / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D010957:Plasmids / Q000235:genetics; D014162:Transfection
D003062:Codon; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; C102997:Msh6 protein, mouse; D004247:DNA