Incorporation of gemcitabine and cytarabine into DNA by DNA polymerase β and ligase III/XRCC1

A. S. Prakasha Gowda, Joanna M. Polizzi, Kristin A. Eckert, Thomas E. Spratt

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

1-β-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 β 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 β 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 kpol/K d for incorporation was dCTP > araCTP, dFdCTP ≫ rCTP. The relative kpol/Kd for FdCTP depended on sequence. The effect on kpol/Kd 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.

Original languageEnglish (US)
Pages (from-to)4833-4840
Number of pages8
JournalBiochemistry
Volume49
Issue number23
DOIs
StatePublished - Jun 15 2010

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gemcitabine
DNA Polymerase III
DNA Ligases
Cytarabine
DNA-Directed DNA Polymerase
Oxidative stress
Ligases
Ligation
Tumors
DNA
Oxidative Stress
Nucleotides
Cytidine
Neoplasms
Deoxycytidine
Chemotherapy
Nucleosides
DNA Repair
Repair
Genes

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Incorporation of gemcitabine and cytarabine into DNA by DNA polymerase β and ligase III/XRCC1",
abstract = "1-β-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 β 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 β 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 kpol/K d for incorporation was dCTP > araCTP, dFdCTP ≫ rCTP. The relative kpol/Kd for FdCTP depended on sequence. The effect on kpol/Kd 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.",
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Incorporation of gemcitabine and cytarabine into DNA by DNA polymerase β and ligase III/XRCC1. / Prakasha Gowda, A. S.; Polizzi, Joanna M.; Eckert, Kristin A.; Spratt, Thomas E.

In: Biochemistry, Vol. 49, No. 23, 15.06.2010, p. 4833-4840.

Research output: Contribution to journalArticle

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AU - Prakasha Gowda, A. S.

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AU - Spratt, Thomas E.

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AB - 1-β-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 β 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 β 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 kpol/K d for incorporation was dCTP > araCTP, dFdCTP ≫ rCTP. The relative kpol/Kd for FdCTP depended on sequence. The effect on kpol/Kd 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.

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