%0 Journal Article
%T Recognition and binding of mismatch repair proteins at an oncogenic hot spot
%A Michael Edelbrock
%A Huiling He
%A Allen Schroering
%A Martha Fernstrom
%A Sangeetha Bathala
%A Kandace J Williams
%J BMC Molecular Biology
%D 2005
%I BioMed Central
%R 10.1186/1471-2199-6-6
%X Electrophoretic mobility shift (gel shift) experiments demonstrate that DNA containing mismatched bases are recognized and bound equally efficiently by hMutS¦Á in both MMR proficient and MMR deficient (hMLH1-/-) nuclear extracts. Competition experiments demonstrate that while hMutS¦Á predictably binds the G:T mismatch to a much greater extent than G:A, hMutS¦Á 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 ¡ú G:C is significantly higher (96%) than that of G:A ¡ú G:C (60%).Combined, these results suggest that initiation of correct MMR requires the contribution of two separate steps; initial recognition by hMutS¦Á 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 hMutS¦Á 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 ¡ú GTC (Gly ¡ú Val) ras-activating mutation found in a high percentage of human tumors.Several different DNA repair systems have evolved within all living cells to correct mispaired or damaged nucleotide residues generated either by endogenous events or by exposure to exogenous mutagenic agents [1,2]. The frequency of mutational events varies widely within the genome, and specific sites harboring increased frequency of mutation are now defined as 'hot spots' of mutation. The human ras protooncogene family contains three such hot spots ¨C codons 12, 13, and 61. Factors contributing to these and other hot spots of mutation are still largely unknown, d
%U http://www.biomedcentral.com/1471-2199/6/6