The fidelity of DNA replication is under constant threat from the formation of lesions within the genome. Oxidation of DNA bases leads to the formation of altered DNA bases such as 8-oxo-7,8-dihydroguanine, commonly called 8-oxoG, and 2-hydroxyadenenine, or 2-OHA. In this work we have examined the incorporation kinetics opposite these two oxidatively derived lesions as well as an abasic site analog by the replicative DNA polymerase from bacteriophage RB69. We compared the kinetic parameters for both wild type and the low fidelity L561A variant. While nucleotide incorporation rates (k pol ) were generally higher for the variant, the presence of a lesion in the templating position reduced the ability of both the wild type and variant DNA polymerases to form ternary enzyme-DNA-dNTP complexes. Thus, the L561A substitution does not significantly affect the ability of the RB69 DNA polymerase to recognize damaged DNA; instead the mutation increases the probability that nucleotide incorporation will occur. We have also solved the crystal structure of the L561A variant forming an 8-oxoG•dATP mispair and show that the propensity for forming this mispair depends on an enlarged polymerase active site.Most replicative DNA polymerases copy DNA with high fidelity. These faithful enzymes must select the correct dNTP among the four canonical nucleotides and do so at rates high enough to support replication of the genome within the time frame of a single cell division. The error rates of replicative DNA polymerases range as high as one error for every 10 6 incorporation events (1). While DNA polymerases have the ability to efficiently discriminate between correct and incorrect incoming nucleoside triphosphates, the genomes of all organisms are under constant assault from both endogenous and exogenous sources. The resulting alterations to the genome can have a dramatic effect on the ability of DNA polymerases to maintain accurate DNA replication or to maintain any replication at all (2). Abasic sites are produced at a rate of >10,000 per human cell per day (3). These lesions arise primarily through spontaneous hydrolysis of the N-glycosylic bond and as intermediates in the base excision repair pathway (4). Abasic sites (Figure 1) are strong blocks to DNA replication by most replicative DNA polymerases and can only be bypassed at a low rate in vitro in the presence of high dNTP concentrations. Under these conditions, all replicative polymerases tested to date exhibit a strong preference for incorporation of dAMP, regardless of the nature of the original templating base. This phenomenon, known as the A-rule, is mutagenic (5,6).* to whom correspondence should be addressed swallace@uvm.edu or sdoublie@uvm.edu tel: 802-656-2164 fax: 802-656-8749. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 March 23. Published in final edited form as:Biochemistry. Reactive oxygen species pose another significant threat to DNA. In particular, 1 is a commonly formed oxidatively derived lesion that has ...

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