Influence of DNA Structure on DNA Polymerase β Active Site Function: EXTENSION OF MUTAGENIC DNA INTERMEDIATES

William A Beard,David D Shock,Samuel H Wilson

doi:10.1074/jbc.m404016200

Abstract

In the ternary substrate complex of DNA polymerase (pol) beta, the nascent base pair (templating and incoming nucleotides) is sandwiched between the duplex DNA terminus and polymerase. To probe molecular interactions in the dNTP-binding pocket, we analyzed the kinetic behavior of wild-type pol beta on modified DNA substrates that alter the structure of the DNA terminus and represent mutagenic intermediates. The DNA substrates were modified to 1) alter the sequence of the duplex terminus (matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. The results indicate that the nucleotide insertion efficiency (k(cat)/K(m), dGTP-dC) is highly dependent on the sequence identity of the matched (i.e. Watson-Crick base pair) DNA terminus (template/primer, G/C approximately A/T > T/A approximately C/G). Mismatches at the primer terminus strongly diminish correct nucleotide insertion efficiency but do not affect DNA binding affinity. Transition intermediates are generally extended more easily than transversions. Most mismatched primer termini decrease the rate of insertion and binding affinity of the incoming nucleotide. In contrast, the loss of catalytic efficiency with homopurine mismatches at the duplex DNA terminus is entirely due to the inability to insert the incoming nucleotide, since K(d)((dGTP)) is not affected. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand.

Highlights

From the Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
The results indicate that the nucleotide insertion efficiency is highly dependent on the sequence identity of the matched (i.e. WatsonCrick base pair) DNA terminus
It provides the polymerase the ability to assess whether geometrical constraints imposed by correct Watson-Crick hydrogen bonding occur, and second, it discourages the templating base from prematurely entering the polymerase active site, which could result in the downstream template base coding for nucleotide insertion [4]

Summary

Introduction

From the Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709. The DNA substrates were modified to 1) alter the sequence of the duplex terminus (matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand. To accurately replicate DNA, polymerases need to stabilize the coding templating base as well as the correct, but not incorrect, incoming nucleotide [5] This is achieved through a series of protein- and substrateinduced conformational changes that result in a dNTP-binding pocket formed by the templating base, DNA duplex terminus, and enzyme. The constraints imposed by the dNTP-binding pocket are determined by DNA sequence (i.e. structure) as well as the conformational fluctuations that occur in response to enzyme and substrate binding

Results

Discussion

Conclusion