Stereochemical Dependence for dSp

1653 WordsJan 28, 20187 Pages
Interestingly, studies from our laboratory and others have demonstrated a stereochemical dependence for dSp processing by enzymes in ODNs. The first instance of a dSp diastereomer difference was reported from our laboratory when Klenow Fragment (exo-) was allowed to insert dATP opposite the isomers for which dSp1-containing ODNs were found to be the more favorable substrate for insertion and bypass.32 This study was extended to in vivo experiments by Neeley, et al. to include pol II and pol IV, also showing more favorable bypass kinetics for dSp1.34 As further evidence for in vivo stereochemical dependency in dSp processing, Henderson, et al. utilized the restriction endonuclease and postlabeling assay (REAP) to determine the mutation profiles for dSp1 and dSp2 in Escherichia coli.28 In the sequence context 5’-d(GSpA)-3’, they found a strong stereochemical dependence in the relative amounts of dG→dT and dG→dC transversion mutations for dSp1 and dSp2. In subsequent experiments, Delaney, et al. showed a somewhat different dependence of mutation profiles on the dSp diastereomers in the sequence context 5’-(TSpG)-3’ using the REAP and competitive replication of adduct bypass assays (CRAB).29,30 An additional observation from our laboratory quantified the digestion kinetics of dSp isomers in the dinucleotides 5’-d(Np[Sp])-3’ (N = A, T, G or C) by nuclease P1 that showed dSp1 was the kinetically preferred substrate in all sequence combinations.33 Lastly, in collaboration

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