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

Abstract

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 hMutSalpha in both MMR proficient and MMR deficient (hMLH1-/-) nuclear extracts. Competition experiments demonstrate that while hMutSalpha predictably binds the G:T mismatch to a much greater extent than G:A, hMutSalpha 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 hMutSalpha 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 hMutSalpha 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.

• Authors: Michael Edelbrock, Huiling He, Allen Schroering, Martha Fernstrom, Sangeetha Bathala, Kandace J Williams
• Journal: BMC molecular biology (BMC Mol Biol) Vol. 6 Pg. 6 (Mar 14 2005) ISSN: 1471-2199 [Electronic] England
• PMID: 15766387 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Codon
• DNA-Binding Proteins
• Proteins
• DNA
• ras Proteins

Topics

• Base Pair Mismatch (genetics)
• Cell Line, Tumor
• Codon (genetics)
• DNA (metabolism)
• DNA Repair
• DNA-Binding Proteins (genetics, metabolism)
• Electrophoretic Mobility Shift Assay (methods)
• HCT116 Cells
• Humans
• Models, Biological
• Plasmids (genetics)
• Point Mutation (genetics)
• Proteins (genetics)
• Proto-Oncogenes (genetics)
• ras Proteins (genetics)