The I260Q variant of DNA polymerase beta extends mispaired primer termini due to its increased affinity for deoxynucleotide triphosphate substrates.

DNA Polymerase beta (pol β), a member of the X-family of DNA polymerases, is a key enzyme in base excision repair (BER) (1). The well-characterized BER pathway removes DNA damage that is induced by reactive oxygen species (ROS) and alkylating agents (2, 3). BER is initiated by DNA glycosylase and the type of glycosylase determines the pathway of BER (4). Monofunctional DNA glycosylases such as Alkyladenine DNA glycosylase (AAG), remove base damage and leave an abasic (AP) site. APE1 cuts the DNA 5′ to the AP site, leaving a 3′OH and a 5′dRP. Pol β removes the 5′dRP and fills in the gap. Bifunctional DNA glycosylases such as 8-OxoG DNA glycosylase 1 (OGG1) remove base damage and leave a modified 3′end, which is modified by Apurinic/apyrimidinic endonuclease 1 (APE1) (5) or by polynucleotide kinase (6). In this pathway, pol β fills in a one to six base gap. During long patch BER, pol β is likely to initiate large gap filling by performing strand displacement synthesis (7). Finally, DNA ligase IIIα-XRCC1 seals the nick (8). Approximately 20,000 lesions/cell/day are channeled through the BER pathway (9) highlighting the importance of this pathway for genomic integrity. Pol β synthesizes DNA with error frequencies ranging from < 10−3 to 10−5, suggesting it is a lower fidelity polymerase than replicative DNA polymerases. However, nucleotide misincorporation by pol β is 10–100-fold lower on a 5′-phosphorylated 1 nt gapped DNA substrate, suggesting that this is likely to be the physiological substrate of pol β (10). We use pol β as a model polymerase to gain insight into mechanisms of polymerase fidelity, due to its small size and lack of proofreading activity. In addition, the structure of DNA polymerase beta is well conserved from parasitic protozoans to humans (11, 12). There are several X-ray and NMR structures available for pol β (13–15) that facilitate interpretation of biochemical studies within the context of the polymerase’s structure. Pol β has a 31 kD polymerase domain which is comprised of three subdomains that consist of the palm, the thumb and the fingers. This architecture represents the hand-like motif of polymerases. In addition, pol β also has an N-terminal 8kD domain that houses its dRP lyase activity (16). We have shown that the hydrophobic hinge region is important for catalysis and fidelity (17–21). This hinge is comprised of the F272, 1174, L194, T196, I260, and Y265 residues and where the motion for rotation of the fingers from an open to closed form originates. It is not part of the active site of pol β. In a previous study we demonstrated that the I260Q variant of the hydrophobic hinge is a mutator polymerase (22). We have shown using presteady state kinetics that it is a global misincorporator due to its inability to discriminate dNTP substrates during the binding step of the polymerase catalytic cycle. Misincorporation would be mutagenic upon extension of the mispaired primer terminus. In our current study we have explored the ability of I260Q to extend mispairs and showed that this variant possesses robust mispair extension activity.