Mechanism of inhibition of B family DNA polymerases by N2-([rho]-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate, BuPdGTP: A Dissertation

Abstract

The B family of DNA-dependent DNA polymerases (pols) are uniquely sensitive to inhibition by N2-(p-n-butylphenyl)deoxyguanosine 5'-triphosphate (BuPdGTP). The affinity of members of the B family for BuPdGTP varies greatly, as does the ability of certain members to use the modified nucleotide as a substrate. For example, eukaryotic pol α has high affinity for the nucleotide, but incorporates it into DNA poorly, while T4 pol has lower affinity, but incorporates the nucleotide well. This thesis addresses two questions: 1) What are the amino acid residue(s) that impart sensitivity to BuPdGTP? and 2) is incorporation of BuPdGTP required for inhibition? To answer the first question, molecular modeling was used with the crystal structure of RB69 pol [Wang et al., 1997], an enzyme closely related to T4 pol [Wang et al., 1995]. This modeling identified a structural pocket adjacent to the polymerase active site that could serve as the butylphenyl "receptor". Based upon this modeling, a chimeric T4 pol containing the residues corresponding to the butylphenyl receptor from human pol α was designed and engineered for expression. The chimera was hypothesized to have a pol α-like phenotype with respect to its response to BuPdGTP (higher sensitivity/ lost ability to incorporate). The chimera was found to be unstable during purification, leaving the hypothesis unresolved. To answer the second question, non-substrate derivatives of BuPdGTP were in which the α,β anhydride oxygen of the triphosphate were replaced with either a CH2 or NH. The ability of the latter derivatives to inhibit polymerase activity and to serve as substrates was measured on T4 pol, RB69 pol and human pol α. Both derivatives retained high potency, but were not substrates under the conditions tested. These compounds were potent, selective inhibitors of B family pol that should be useful in the formation of a stable complex of enzyme:DNA:inhibitor for crystallization trials.