The dominant role of proofreading exonuclease activity of replicative polymerase ε in cellular tolerance to cytarabine (Ara-C)
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Masataka Tsuda1, Kazuhiro Terada1, Masato Ooka2, Koji Kobayashi2, Hiroyuki Sasanuma1, Ryo Fujisawa3, Toshiki Tsurimoto3, Junpei Yamamoto4, Shigenori Iwai4, Kei Kadoda1,5, Remi Akagawa1, Shar-Yin Naomi Huang6, Yves Pommier6, Julian E. Sale7, Shunichi Takeda1 and Kouji Hirota1,2
1Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-Ku, Kyoto 606-8501, Japan
2Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Hachioji-Shi, Tokyo 192-0397, Japan
3Department of Biology, School of Sciences, Kyushu University, Nishi-Ku, Fukuoka 819-0395, Japan
4Division of Chemistry, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan
5Division of Radiation Life Science, Research Reactor Institute, Kyoto University, Kumatori, Sennan, Osaka 590-0494, Japan
6Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
7Medical Research Council Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK
Kouji Hirota, email: [email protected]
Shunichi Takeda, email: [email protected]
Keywords: replicative polymerase ε, proofreading exonuclease, chainterminator, nucleoside analog, cytarabine (Ara-C)
Received: January 04, 2017 Accepted: February 28, 2017 Published: March 23, 2017
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.
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