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

Abstract

BackgroundThe 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.ResultsElectrophoretic mobility shift (gel shift) experiments demonstrate that DNA containing mismatched bases are recognized and bound equally efficiently by hMutSα in both MMR proficient and MMR deficient (hMLH1-/-) nuclear extracts. Competition experiments demonstrate that while hMutSα predictably binds the G:T mismatch to a much greater extent than G:A, hMutSα 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%).ConclusionCombined, these results suggest that initiation of correct MMR requires the contribution of two separate steps; initial recognition by hMutSα 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 hMutSα 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.

Genomic Characterization and Phylogenetic Position of Two New Species in Rhabdoviridae Infecting the Parasitic Copepod, Salmon Louse (Lepeophtheirus salmonis)

“…An assay targeting the elongation factor from salmon louse was used as internal control [31] . During real time RT PCR on salmon tissues an assay targeting the elongation factor alpha from Atlantic salmon was used as internal control [32] .…”

Structural, molecular and cellular functions of MSH2 and MSH6 during DNA mismatch repair, damage signaling and other noncanonical activities

“…In addition to ATP binding, all MutS complexes have intrinsic ATPase activity conferred by an A denine nucleotide b inding c assette (ABC) motif, as confirmed by both biochemical and genetic studies [58–60]. Binding of the MutSα complex to a mismatched DNA substrate can be disrupted by the addition of excess ATP, however MutSα -driven hydrolysis of ATP is necessary for in vitro MMR [50, 60, 61]. Additional evidence has been provided by crystallization studies revealing ADP bound by both hMSH2 and hMSH6 in complex with mismatch-containing DNA [32].…”

“…MutSα recognizes specific lesions including O 6 meG, complex pyrimidine dimers, halogenated pyrimidines, bulky adducts such as benzo[ c ]phenanthrene dihydrodiol epoxide, and cisplatin adducts [134–137]. The MutSα complex has different binding affinity and repair efficiency, depending on the specific DNA lesion and sequence context [3, 21, 61, 133, 135,136]. MutSα has higher binding affinity to specifically modified bases that are also mismatched, particularly O 6 meG:T, indicating increased lesion recognition after DNA replication or an error-prone repair attempt.…”

“…[22, 142]. In comparison, repair of G:A and 8-oxoG:A is very inefficient [22, 61, 142]. Binding of MutSα to an 8-oxoG:A mismatch induces ATP hydrolysis and ADP→ ATP exchange, indicating that MutSα recognizes, binds, and is at least partially activated by this lesion despite that it is poorly repaired by MMR [141].…”