Incorporation of gemcitabine and cytarabine into DNA by DNA polymerase beta and ligase III/XRCC1.
1-Beta-D-arabinofuranosylcytosine (cytarabine, araC) and 2',2'-difluoro-2'-deoxycytidine (gemcitabine, dFdC), are effective cancer chemotherapeutic agents due to their ability to become incorporated into DNA and then subsequently inhibit DNA synthesis by replicative DNA polymerases. However, the impact of these 3'-modified nucleotides on the activity of specialized DNA polymerases has not been investigated. The role of polymerase beta and base excision repair may be of particular importance due to the increased oxidative stress in tumors, increased oxidative stress caused by chemotherapy treatment, and the variable amounts of polymerase beta in tumors. Here we directly investigate the incorporation of the 5'-triphosphorylated form of araC, dFdC, 2'-fluoro-2'-deoxycytidine (FdC), and cytidine into two nicked DNA substrates and the subsequent ligation. Opposite template dG, the relative k(pol)/K(d) for incorporation was dCTP > araCTP, dFdCTP >> rCTP. The relative k(pol)/K(d) for FdCTP depended on sequence. The effect on k(pol)/K(d) was due largely to changes in k(pol) with no differences in the affinity of the nucleoside triphosphates to the polymerase. Ligation efficiency by T4 ligase and ligase III/XRCC1 was largely unaffected by the nucleotide analogues. Our results show that BER is capable of incorporating araC and dFdC into the genome.
Prakasha Gowda
A S
AS
Department of Biochemistry and Molecular Biology, The Pennsylvania State University,Hershey, Pennsylvania 17033, USA.
Polizzi
Joanna M
JM
Eckert
Kristin A
KA
Spratt
Thomas E
TE
D000964:Antimetabolites, Antineoplastic / Q000378:metabolism*; D002384:Catalysis; D003561:Cytarabine / Q000378:metabolism*; D004249:DNA Damage / Q000502:physiology*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D019951:DNA Polymerase beta / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D000075223:Poly-ADP-Ribose Binding Proteins; D015398:Signal Transduction / Q000502:physiology; D013379:Substrate Specificity; D000076105:X-ray Repair Cross Complementing Protein 1; D029867:Xenopus Proteins; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D029867:Xenopus Proteins; D003561:Cytarabine; D003841:Deoxycytidine; D019951:DNA Polymerase beta; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618817:LIG3 protein, human; D000093542:Gemcitabine