The Asp285 variant of DNA polymerase beta extends mispaired primer termini via increased nucleotide binding.

Endogenous DNA damage occurs at a rate of at least 20,000 lesions per cell per day (2). These endogenous lesions are repaired by the base excision repair (BER) machinery, and their correct repair is critical for genome stability. A key enzyme in the BER pathway is DNA polymerase beta (polβ), which removes the deoxyribose phosphate group (dRP) and fills in the gap with a nucleotide after the DNA lesion is excised. A relatively error-prone polymerase, pol β inserts an incorrect nucleotide in approximately one out of every 10,000 nucleotide insertions (3). Given that BER is responsible for repairing about 20,000 DNA lesions per cell per day, pol β could potentially incorporate incorrect nucleotides on the order of one per cell per day. These errors in DNA repair can be carried through further cell divisions, which can increase genomic instability. Increased genomic instability has been shown to lead to the generation of a mutator phenotype, which in turn can produce a malignant phenotype (4). In BER, damaged DNA bases are recognized and excised by a specific DNA glycosylase, which generates a mutagenic apurinic/apyrimidinic (AP) site (5). Next, AP Endonuclease 1 incises the DNA backbone on the 3′ side of the AP site, leaving behind a dRP group (6), which is then removed by pol β. Pol β also fills in the single-base gap in the DNA with the correct nucleotide. Lastly, DNA Ligase IIIα seals the nick in the backbone to complete the repair. Pol β is a 39kDa protein, which has two catalytic functions: it catalyzes the synthesis of DNA and the removal of dRP groups from DNA (7). Pol β does not possess any proofreading exonuclease activity, unlike other eukaryotic polymerases such as pol δ and pol e (8). Pol β functions both in BER and in meiosis (9), and additionally, pol β variants have been found in several types of cancerous tissue (3). Therefore, studying pol β and its variants can lead to a deeper understanding of the mutational basis of cancer (10). Synthesis of DNA can be abated by the nucleoside analog drug 3′-azido-3′deoxythymidine (AZT). AZT can be incorporated into DNA, but it causes chain termination via the azido group in the 3′ position, which is unable to be extended by the polymerase (1). Using an in vivo selection, several pol β variants were identified as conferring resistance to AZT to E. coli cells (1). Three of these variants have already been studied in greater detail in vitro (Figure 1a): D246V,(1, 11) R253M,(1) and E249K (12). The Val246 variant was shown to misincorporate nucleotides through altered DNA positioning in the active site (11). The Lys249 variant was shown to be an extender of mispaired primer termini without alteration of the misincorporation fidelity (12). The Met253 variant exhibits increased catalytic efficiency for incorporation of dTTP while at the same time showing a marked reduction in incorporation of AZT when compared to wild-type (1). Figure 1 (a) Overall structure of pol β complexed with gapped DNA (29) in the fully closed conformation colored according to subdomains (8Kd, red; thumb, green; palm, magenta; fingers, blue; 1bpy). Residues Asp246, Glu249, Arg253 and His285, all of which ... The goal of the work presented herein is to examine in detail a fourth AZT resistant mutant of pol β -- the His285 to Asp variant. His285 is located on Helix N in the fingers subdomain of pol β, as shown in Figure 1b. Helix N encompasses residues 275 through 289, which includes several residues that are critical for pol β fidelity. Helix N in pol β contacts the DNA along the minor groove, when the polymerase is in the closed conformation (bound to both DNA and dNTP), and allows for formation of the dNTP binding pocket. Due to altered DNA positioning, the Lys289 to Met variant shows increased misincorporation of dCTP and dGTP opposite template C and G, respectively, and K289M has been found in colorectal carcinomas (13). Alteration of Met282 to Leu causes an increase in the overall stability of pol β resulting in a reduction in fidelity due to poorer dNTP discrimination at the level of ground state binding (Kd(dNTP)) (14). Two other important residues reside on Helix N, Asp276 and Arg283 (not shown). Arg283 contacts the minor groove of the DNA substrate on the template side, and its alteration to Ala or Lys results in both deletion and base substitution mutations (15). Asp276 when changed to Val allows the polymerase to extend mispaired primer termini without a significant increase in misincorporation events (16). His285 is located near the C-terminal end of Helix N and comes in close contact with both Lys289 and Trp325 (Figure 1c). All of these structural clues suggest that mutation of His285 to Asp could indirectly impact the fidelity of pol β. It is presented herein that like the D276V variant, pol β H285D increases the extension frequency of mispaired primer termini without an increase in misincorporation. Through single-turnover kinetics it is shown that this increase in mispair extension is due to a dramatic increase in nucleotide binding when compared to wild-type pol β.