Chemotherapeutics and DNA polymerase beta mechanism of modified nucleotide discrimination

Nucleoside analogs like cytosine arabinoside (araC) and cytosine 2', 2'-difluorodeoxyriboside (gemcitabine; dFdC) are the paradigms of anticancer therapy. AraC is the single most effective agent against adult acute leukemia, while gemcitabine is the drug of choice for multitudes solid tumors. Although thousands of patients are exposed to these nucleoside analogs regularly, there is limited knowledge of the molecular mechanisms of these agents. Our study uses mammalian DNA polymerase beta to study the mechanism of action of such drugs. Since incorporation of these analogs by DNA polymerases is critical for optimal antitumor activity, a better understanding of nucleoside discrimination by DNA polymerases is essential for novel anticancer drug design to maximize response/efficacy and minimize resistance/toxicity.; Our project uses a structure-function approach to investigate the mechanism of nucleotide (dNTP) analog discrimination by DNA pol beta. The analogs investigated were araCTP, dFdCTP, AZT-TP, 2'-fluorodeoxyribonucleotide triphosphate (2'FdCTP), and O6-methyldeoxyguanosine triphosphate (m6GTP). The pol beta variants studied were located in the active site (Tyr-271), and the loop region of the palm sub-domain (Glu-249, Pro-242 and Arg-253). Discrimination of incoming dNTPs by pol beta wild-type (WT) and variants was examined using the in vitro HSV-thymidine kinase forward mutation assay, and steady-state kinetic analyses.; We hypothesize that pol beta active site position 271 has a steric mechanism for discriminating sugar-modified nucleotide analogs. Position 253 at the junction of the loop region may function as a structural 'gate' for incoming nucleotides. The higher the rigidity of this junction, the better the polymerase becomes at discriminating modified nucleotides. The naturally occurring polymorphism P242R behaves similarly to WT when discriminating sugar-modified dNTPs. In addition, positions Glu-249 and Arg-253 may be important for base-modified nucleotide discrimination.; Overall, we provide evidence that a single amino acid change within the pol beta active site may be responsible for dNTP verses rNTP discrimination, and residues located in the distant loop region may be involved in channeling dNTPs moving toward the active site. In-depth structure-function analyses such as those presented here may provide a basis for improved chemotherapeutic drug design geared toward improved drug incorporation into DNA and enhanced therapeutic efficacy.