The dominant role of proofreading exonuclease activity of replicative polymerase ε in cellular tolerance to cytarabine (Ara-C)

Masataka Tsuda, Kazuhiro Terada, Masato Ooka, Koji Kobayashi, Hiroyuki Sasanuma, Ryo Fujisawa, Toshiki Tsurimoto, Junpei Yamamoto, Shigenori Iwai, Kei Kadoda, Remi Akagawa, Shar Yin Naomi Huang, Yves Pommier, Julian E. Sale, Shunichi Takeda, Kouji Hirota

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

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. γH2AX focus formation following a pulse of Ara-C was immediate and did not progress into the next round of replication, while γH2AX 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-exoholoenzyme 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.

Original languageEnglish
Pages (from-to)33457-33474
Number of pages18
JournalOncotarget
Volume8
Issue number20
DOIs
Publication statusPublished - 2017

All Science Journal Classification (ASJC) codes

  • Oncology

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