Chemotherapeutics and DNA Polymerase Beta Mechanism of Modified Nucleotide Discrimination
Open Access
- Author:
- Hamid, Subarna
- Graduate Program:
- Biochemistry and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 10, 2005
- Committee Members:
- Kristin Ann Eckert, Committee Chair/Co-Chair
Neil David Christensen, Committee Member
Momcilo Miljkovic, Committee Member
Edward Joseph Gunther, Committee Member
Judith S Bond, Committee Member - Keywords:
- AraC
Gemcitabine
Chemotherapeutics
Nucleoside analogs
DNA Polymerase
DNA Polymerase Beta
Steady State Kinetic Analysis
Mutation Frequency - Abstract:
- Nucleoside analogs like cytosine arabinoside (araC) and cytosine 2’, 2’-difluorodeoxyriboside (gemcitabine) are the paradigms of anticancer therapy. AraC is the single most effective agent for treatment of adult acute leukemia, while gemcitabine is the drug of choice for combination therapy of multitudes of solid tumors including those of the lung, pancreas, ovary and breast. Although thousands of patients are exposed to these nucleoside analogues regularly, there is limited knowledge of the molecular mechanisms of these agents. Our study uses the mammalian DNA polymerase ? to study the mechanism of action of such nucleoside analogues. A better understanding of nucleoside analogue discrimination by DNA polymerases is essential for novel anticancer drug design to maximize response/efficacy and minimize resistance/toxicity. DNA polymerase ? (pol ?) was used as a model system to study the mechanism of nucleoside analogues AraC, Gemcitabine, and 3’-Azido-deoxythymidine (AZT). AZT is an antiviral agent, while AraC and Gemcitabine are widely used for the treatment of human cancers. All three must be incorporated into the nascent DNA strand by DNA polymerases for optimal anti-viral/anti-tumor activity. Our data clearly indicates that 3’-modified nucleotide analogs, such as AZT-TP, are much more strongly discriminated against compared to 2’-modified nucleotide analogs, like araC triphosphate and gemcitabine triphosphate. Chemotherapeutic nucleoside analogues are often administered in combination with alkylating agents that can result in up-regulation of DNA repair enzymes such as pol ?. This project used a structure-function approach to investigate the mechanism of nucleotide (dNTP) analogue discrimination by DNA pol ?. The variants studied are located in the active site (Tyr-271), and the loop region of the palm sub-domain (Glu-249, Arg-253 and Pro-242). Discrimination of incoming nucleotides by these variants was examined using the in vitro HSV-thymidine kinase forward mutation assay, and steady-state kinetic analyses. Recombinant pol ? active site variants Y271L and Y271G were purified from E. coli and shown to have a similar enzymatic activity with dCTP relative to wild type (WT) pol ?. However the variants show a 6 to 10-fold (Y271L), and 37-fold (Y271G) decrease in inherent reactivity (Vmax/Km) ratios with sugar modified dNTPs. The similar overall activity of the variants Y271L and Y271G with natural dCTP indicates that position 271 is not critical for the general conformation/catalysis of the enzyme. We postulate that the change of the electronegative and bulky Tyr at 271 caused the dramatic decrease in inherent reactivity ratios with modified dNTPs. When a dCTP:rCTP competition reaction was performed with Y271G, the variant demonstrated a complete loss of discrimination between dCTP and rCTP, relative to WT. This strongly suggests that loss of electronegativity at 271 results in lower dNTP discrimination (Y271L), but the additional loss of size (Y271G) completely eliminates discrimination of sugar-modified dNTPs. Other family X polymerases, pol ? and ?, have a highly conserved glycine at a position homologous to pol ? 271. Residue Arg-253 lies at the junction of the 240-253-loop region of the palm sub-domain of pol ?. R253M is known to confer an AZT-resistant in vivo phenotype, and we confirmed this in vitro by steady-state kinetic analysis. To determine the function of position 253 in dNTP discrimination, we constructed and purified recombinant R253G, R253C and R253K. Pol ? variant R253G showed extremely low catalytic activity, implying that size plays an important role in stabilizing the junction of the loop. Variants R253M, R253C and R253K were 5-, 50 and 100-fold less catalytically active, respectively, with natural dCTP, relative to WT. The discrimination at the level of Km for rCTP incorporation by R253M and R253C were 2 to 3-fold higher than to WT, while that of R253K was 3- fold lower. Based on these results and computer modeling, we propose that the level of rigidity within the junction of the loop may be critical for incoming nucleotide discrimination by DNA pol ?. In addition, loop variants E249K and P242R were also examined. P242R is the only naturally occurring, coding region pol ? polymorphism identified to date in human populations. This is the first characterization of this variant to the best of our knowledge. We determined that Arg-242 behaves similarly to WT when discriminating AraCTP and 2’FdCTP, implying that the flexible arginine side-chain may be held in a rigid conformation, mimicking the proline imine ring, in position 242. The mutation specificity for P242R was also identical to WT. Therefore, we hypothesize that patients with this polymorphism may not have any unusual phenotypes when treated with sugar-modified nucleotide analogues. We also established that the pol ? residues Arg-253 and Glu-249 are involved in base-modified nucleotide discrimination, implying that regions far from the active site may play a role in modified nucleotide incorporation within the DNA. We conclude that pol ? active site position 271 has an electrostatic and steric mechanism for discriminating sugar-modified nucleotide analogues such as AraC and Gemcitabine. In addition, 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 (due to a combination of hydrogen bonds and sulfide bridges), the better the polymerase becomes at discriminating modified nucleotides. 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.