Recognition and binding of mismatch repair proteins at an oncogenic hot spot

Background: The current investigation was undertaken to determine key steps differentiating G: T and G: A repair at the H-ras oncogenic hot spot within the nuclear environment because of the large difference in repair efficiency of these two mismatches. Results: Electrophoretic mobility shift ( gel shift) experiments demonstrate that DNA containing mismatched bases are recognized and bound equally efficiently by hMutS alpha in both MMR proficient and MMR deficient ( hMLHI-/-) nuclear extracts. Competition experiments demonstrate that while hMutSa predictably binds the G: T mismatch to a much greater extent than G: A, hMutSa demonstrates a surprisingly equal ratio of competitive inhibition for both G: T and G: A mismatch binding reactions at the H-ras hot spot of mutation. Further, mismatch repair assays reveal almost 2-fold higher efficiency of overall G: A repair (5'-nick directed correct MMR to G: C and incorrect repair to T: A), as compared to G: T overall repair. Conversely, correct MMR of G: T. G: C is significantly higher (96%) than that of G: A -> G: C (60%). Conclusion: Combined, these results suggest that initiation of correct MMR requires the contribution of two separate steps; initial recognition by hMutSa followed by subsequent binding. The 'avidity' of the binding step determines the extent of MMR pathway activation, or the activation of a different cellular pathway. Thus, initial recognition by hMutSa in combination with subsequent decreased binding to the G: A mismatch ( as compared to G: T) may contribute to the observed increased frequency of incorrect repair of G: A, resulting in the predominant GGC -> GTC (Gly -> Val) ras-activating mutation found in a high percentage of human tumors.