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Hogg, Matthew

Cell and Molecular Biology Program

2005

Ph. D.

The driving force behind all life is the requirement to reproduce and allow the existence of the species to propagate through the continuum of time. As all information telling an organism to be the way it is resides in its genetic code, reproduction requires that the genetic code be copied. And copied correctly lest mistakes accumulate to the point where the organism is no longer viable. All life, therefore, has the ability to replicate its genetic material in an efficient and accurate manner through the use of complicated arrangements of molecular machines, collectively known as the replisome. At the core of the replisome is the DNA polymerase, the enzyme directly responsible for the accurate incorporation of new DNA bases opposite the original template. This enzyme, along with other proteins in the replisome, can copy DNA at the rates of several hundred bases per second and with error levels approaching 1 mistake for every 100,000,000 correct base pairs formed. But the genome of every organism is under constant assault from environmental and metabolic agents that can alter the physical character of the genetic material. Arguably the most common lesions appearing in DNA are abasic sites where the coding nucleotide has been lost. Replicative polymerases tend to stall at these sites and, if they do make it past, have a high probability of inserting the wrong nucleotide opposite the abasic site. Either situation is detrimental to the cell, the first leading to apoptosis and the second leading to mutagenesis. In this work we have characterized the interactions of the replicative DNA polymerase from bacteriophage RB69 with primer/template DNA upon incorporation of an A opposite a stable abasic site analog. We show, by crystallizing the enzyme/DNA complex and capturing three distinct structural conformations, that upon addition of the nucleotide opposite the abasic site the enzyme fails to translocate past the abasic site. Instead the duplex DNA becomes distorted, pulls away from the polymerase active site, and then the primer end dissociates from the template and switches into the exonuclease active site. Kinetic analyses of the incorporation rates of nucleotides opposite abasic sites suggest that exonuclease deficient polymerase mutants exhibit altered polymerization rates and that homologous enzymes from bacteriophage RB69 and bacteriophage T4 show altered abilities to make mistakes when challenged with abasic sites or base pair mismatches.