title: The dominant role of proofreading exonuclease activity of replicative polymerase ε in cellular tolerance to cytarabine (Ara-C).
abstract: Chemotherapeutic nucleoside analogs, such as Ara-C, 5-Fluorouracil (5-FU) and Trifluridine (FTD), are frequently incorporated into DNA by the replicative DNA polymerases. However, it remains unclear how this incorporation kills cycling cells. There are two possibilities: Nucleoside analog triphosphates inhibit the replicative DNA polymerases, and/or nucleotide analogs mis-incorporated into genomic DNA interfere with the next round of DNA synthesis as replicative DNA polymerases recognize them as template DNA lesions, arresting synthesis. To address the first possibility, we selectively disrupted the proofreading exonuclease activity of DNA polymerase ε (Polε), the leading-strand replicative polymerase in avian DT40 and human TK6 cell lines. To address the second, we disrupted RAD18, a gene involved in translesion DNA synthesis, a mechanism that relieves stalled replication. Strikingly, POLE1exo-/- cells, but not RAD18-/- cells, were hypersensitive to Ara-C, while RAD18-/- cells were hypersensitive to FTD. gH2AX focus formation following a pulse of Ara-C was immediate and did not progress into the next round of replication, while gH2AX focus formation following a pulse of 5-FU and FTD was delayed to the next round of replication. Biochemical studies indicate that human proofreading-deficient Polε-exo- holoenzyme incorporates Ara-CTP, but subsequently extend from this base several times less efficiently than from intact nucleotides. Together our results suggest that Ara-C acts by blocking extension of the nascent DNA strand and is counteracted by the proofreading activity of Polε, while 5-FU and FTD are efficiently incorporated but act as replication fork blocks in the subsequent S phase, which is counteracted by translesion synthesis.
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-Ku, Kyoto 606-8501, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Tsuda
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-Ku, Kyoto 606-8501, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Terada
authors.affiliation: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Hachioji-Shi, Tokyo 192-0397, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Ooka
authors.affiliation: Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Hachioji-Shi, Tokyo 192-0397, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-Ku, Kyoto 606-8501, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Sasanuma
authors.affiliation: Department of Biology, School of Sciences, Kyushu University, Nishi-Ku, Fukuoka 819-0395, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Fujisawa
authors.affiliation: Department of Biology, School of Sciences, Kyushu University, Nishi-Ku, Fukuoka 819-0395, Japan.
authors.forename: Toshiki
authors.initials: T
authors.lastname: Tsurimoto
authors.affiliation: Division of Chemistry, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan.
authors.forename: Junpei
authors.initials: J
authors.lastname: Yamamoto
authors.affiliation: Division of Chemistry, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan.
authors.forename: Shigenori
authors.initials: S
authors.lastname: Iwai
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-Ku, Kyoto 606-8501, Japan.|Division of Radiation Life Science, Research Reactor Institute, Kyoto University, Kumatori, Sennan, Osaka 590-0494, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Kadoda
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-Ku, Kyoto 606-8501, Japan.
authors.forename: Remi
authors.initials: R
authors.lastname: Akagawa
authors.affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Shar-Yin Naomi
authors.initials: SN
authors.lastname: Huang
authors.affiliation: Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Yves
authors.initials: Y
authors.lastname: Pommier
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK.
authors.forename: Julian E
authors.initials: JE
authors.lastname: Sale
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-Ku, Kyoto 606-8501, Japan.
authors.forename: Shunichi
authors.initials: S
authors.lastname: Takeda
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-Ku, Kyoto 606-8501, Japan.|Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Hachioji-Shi, Tokyo 192-0397, Japan.
authors.forename: Kouji
authors.initials: K
authors.lastname: Hirota
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D002453:Cell Cycle / Q000235:genetics; D045744:Cell Line, Tumor; D003561:Cytarabine / Q000494:pharmacology*; D004249:DNA Damage; D004257:DNA Polymerase II / Q000235:genetics* / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics; D004361:Drug Tolerance / Q000235:genetics*; D055786:Gene Knockout Techniques; D005838:Genotype; D006801:Humans; D009154:Mutation; D044767:Ubiquitin-Protein Ligases / Q000235:genetics
chemical_list: D000964:Antimetabolites, Antineoplastic; D004268:DNA-Binding Proteins; C485954:RAD18 protein, human; D003561:Cytarabine; D044767:Ubiquitin-Protein Ligases; D004257:DNA Polymerase II
keywords: chainterminator; cytarabine (Ara-C); nucleoside analog; proofreading exonuclease; replicative polymerase ε