Proofreading exonuclease activity of replicative polymerase epsilon promotes cellular tolerance to arabinosides in CTF18-dependent and -independent manner

Abstract

Ara-A, Ara-C, Ara-G, and Ara-T are arabinose sugars combined with adenine, cytosine, guanine, and thymine bases, respectively. These drugs are clinically important as these drugs are commonly used as anti-viral and anti-cancer drugs. Ara-C, an arabinoside, serves as a chain terminator of deoxyribonucleic acid (DNA) replication by interfering with replication after it is incorporated at the 3′ end of nascent DNA, thereby restricting the proliferation of viruses and cancer cells. The incorporated Ara-CMP is efficiently removed by the proofreading exonuclease activity of polymerase epsilon (Polε), in which the alternative clamp loader CTF18 plays a pivotal role. However, the requirement of CTF18 for the removal of the other arabinosides from the 3′ end of nascent DNA remains unclear. Here, we explored DNA repair pathways responsible for the cellular tolerance to Ara-A and found that cells deficient in the proofreading exonuclease activity of Polε (POLE1exo−/−) showed the highest sensitivity to Ara-A. This activity was also required for cellular tolerance to Ara-G and Ara-T. CTF18−/− cells showed higher Ara-A sensitivity than wild-type cells, though it was critically lower than that of POLE1exo−/− cells. Similar trends were observed for the sensitivity to Ara-G and Ara-T. These results indicate that these arabinosides are removed by Polε proofreading exonuclease activity, and CTF18 is pivotal for Polε-mediated Ara-C removal but does not play critical roles for Polε-mediated removal of Ara-A, Ara-G, and Ara-T. In this study, we unveiled a difference between Ara-C and the other arabinosides (Ara-A, Ara-G, and Ara-T) in the removal from the 3′ end of nascent DNA.