Abstract:
Deficiencies in DNA mismatch repair have been found in hereditary cancers as well as in sporadic cancers, illustrating the importance of mismatch repair in maintaining genomic integrity. To determine if inefficient mismatch repair can contribute to hotspots of mutation, repair rates were determined 'in vivo' in mammalian cells for mismatched nucleotides located at H-ras codon 10 and compared to previously determined repair rates at a nearby activating hotspot of mutation, H-ras codon 12. Repair rates for H-ras codon 10 are significantly improved over repair rates at codon 12. This indicates that inefficiencies in mismatch repair are responsible, at least in part, for the well-documented hotspot of mutation at codon 12 and that surrounding sequence context can effect repair of mismatches. Gel-shift analysis demonstrates that the degree of binding by the initial mismatch recognition factor hMutS[alpha] (heterodimer of hMSH6 and hMSH2) correlates with 'in vivo' repair rates for each mismatch tested at the codon 12 location. UV cross-linking of nuclear proteins to G:A and G:T mismatches at codon 10 or codon 12 generally confirm these results. Overall this suggests that there is lowered efficiency in the kinetics of mismatch repair at codon 12, perhaps in the initiation step, rather than innaccurate repair leading to mutation. The interactions of specific mismatch repair proteins in human nuclear extracts were then examined to determine the proteins binding to mismatched DNA. Immunoprecipitation followed by Western blotting indicates two novel complexes that exist in the absence of ATP: one consisting of hMSH2, hMSH6, hMLH1 and hPMS2 and the other consisting of hMSH2, hMSH6, hMLH1 and hPMS1. Furthermore, the protein complexes specifically bind to mismatched DNA and not to a similar homoduplex oligonucleotide. The protein complex-DNA interactions occur primarily through hMSH6, although hMSH2 can also become cross-linked to the mismatched substrate. In the presence of ATP, the binding of hMSH6 to mismatched DNA is decreased. In addition, hMLH1, hPMS2 and hPMS1 no longer interact with each other or with the hMutS[alpha] complex. However, the ability of hMLH1 to co-immunoprecipitate mismatched DNA 'increases' in the presence of ATP, suggesting a role for hMLH1 in subsequent ATP-dependent repair processes.
