The dominant role of proofreading exonuclease activity of replicative polymerase ε in cellular tolerance to cytarabine (Ara-C)
| Autor: | Hiroyuki Sasanuma, Koji Kobayashi, Toshiki Tsurimoto, Ryo Fujisawa, Shunichi Takeda, Kei Kadoda, Masato Ooka, Shar Yin Naomi Huang, Kazuhiro Terada, Kouji Hirota, Junpei Yamamoto, Julian E. Sale, Shigenori Iwai, Remi Akagawa, Masataka Tsuda, Yves Pommier |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Exonuclease DNA Replication Antimetabolites Antineoplastic Genotype DNA polymerase proofreading exonuclease Ubiquitin-Protein Ligases cytarabine (Ara-C) 03 medical and health sciences chemistry.chemical_compound Gene Knockout Techniques 0302 clinical medicine Cell Line Tumor chainterminator Humans Gene Polymerase Genetics biology DNA synthesis Cell Cycle nucleoside analog Cytarabine replicative polymerase ε DNA Polymerase II Drug Tolerance Virology 3. Good health DNA-Binding Proteins genomic DNA 030104 developmental biology Oncology chemistry 030220 oncology & carcinogenesis Mutation biology.protein Proofreading DNA Research Paper DNA Damage |
| Zdroj: | Oncotarget |
| ISSN: | 1949-2553 |
| Popis: | // Masataka Tsuda 1 , Kazuhiro Terada 1 , Masato Ooka 2 , Koji Kobayashi 2 , Hiroyuki Sasanuma 1 , Ryo Fujisawa 3 , Toshiki Tsurimoto 3 , Junpei Yamamoto 4 , Shigenori Iwai 4 , Kei Kadoda 1, 5 , Remi Akagawa 1 , Shar-Yin Naomi Huang 6 , Yves Pommier 6 , Julian E. Sale 7 , Shunichi Takeda 1 and Kouji Hirota 1, 2 1 Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshidakonoe, Sakyo-Ku, Kyoto 606-8501, Japan 2 Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Hachioji-Shi, Tokyo 192-0397, Japan 3 Department of Biology, School of Sciences, Kyushu University, Nishi-Ku, Fukuoka 819-0395, Japan 4 Division of Chemistry, Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka 560-8531, Japan 5 Division of Radiation Life Science, Research Reactor Institute, Kyoto University, Kumatori, Sennan, Osaka 590-0494, Japan 6 Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA 7 Medical Research Council Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK Correspondence to: Kouji Hirota, email: khirota@tmu.ac.jp Shunichi Takeda, email: stakeda@rg.med.kyoto-u.ac.jp Keywords: replicative polymerase e, proofreading exonuclease, chainterminator, nucleoside analog, cytarabine (Ara-C) Received: January 04, 2017 Accepted: February 28, 2017 Published: March 23, 2017 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 e (Pole), 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, POLE1 exo-/- 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 Pole-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 Pole, 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. |
| Databáze: | OpenAIRE |
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