Faithful replication of genomic DNA is essential for maintaining genomic stability. The high fidelity of eukaryotic replication associated with an error frequency of approximately 10-6–10-8 is accomplished through two mechanisms [1]. First, the constrained polymerase active site of replicative polymerases allows for the incorporation of nucleotides forming correct Watson–Crick base pairings, thus preventing the incorporation of mispaired nucleotides [2], [3]. Second, the 3′–5′ exonuclease activity of replicative polymerases performs proofreading by eliminating misincorporated nucleotides [4], [5]. Polymerase epsilon (Polε) is involved in leading strand synthesis [6], [7] and consists of four subunits, namely, PolE1, PolE2, PolE3, and PolE4, with the PolE1 subunit possessing both polymerase and exonuclease activity [8], [9], [10]. A number of missense mutations at highly conserved residues in the proofreading motif of Polε have been reported in cancer cells, suggesting that proofreading exonuclease activity plays a pivotal role in the suppression of mutagenesis and cancer development [11], [12], [13], [14], [15]. Purified Polε performs proofreading and subsequent DNA synthesis without additional factors [16], [17], [18]. However, it is unclear whether the Polε holoenzyme is sufficient to perform the proofreading in vivo. If not, the next question is which additional factors facilitate proofreading by Polε.

Nucleoside analogs have been used to treat cancers and viral infections. Cytarabine (Ara-C), an analog of cytosine nucleoside, has been used in the treatment of acute lymphocytic and myeloid leukemia [19]. Ara-C is efficiently incorporated into genomic DNA during DNA replication and inhibits DNA synthesis from the incorporated Ara-CMP [18]. The inhibitory effect of Ara-CMP on the purified human Polε holoenzyme at the growing end of the nascent has been reported [18], suggesting that the incorporated Ara-CMP serves as a chain terminator. Despite the inhibitory action of Ara-C on replicative polymerases in vitro [18], large amounts of Ara-CMP are incorporated into genomic DNA [20], [21]. The genomically embedded Ara-CMP might interfere with the progression of replicative polymerases due to local structural distortion. Translesion synthesis (TLS) and homologous recombination (HR) contribute to restarting stalled forks [22], [23], [24], and repliming by PrimPol is also critical for the DNA replication restart [25]. However, unlike other CTNAs, Ara-C selectively sensitizes POLE1D269A/- cells but not cells deficient in HR, TLS, base excision repair, or PrimPol [18]. Thus, Ara-C and POLE1D269A/- cells provided a unique opportunity to identify genes involving the proofreading by Polε, using the epistatic analysis with POLE1D269A/- cells.

CTF18 is a component of the alternative clamp loader complex CTF18-RFC, which is involved in sister chromatid cohesion (SCC) establishment [26], [27], [28], [29]. CTF18 also plays a role in replication, as evidenced by the fact that CTF18 interacts with stalled Polε and efficiently loads the PCNA sliding clamp to restore Polε-mediated synthesis [30], [31], [32], [33]. Moreover, leading strand synthesis by Polε is facilitated through PCNA loading by the clamp loader complex CTF18-RFC in vitro, whrere the Polε-CTF18 interaction is pivotal [34]. However, the roles played by the CTF18 in replication restart at the site of Ara-CMP insertion have not been elucidated.

In this study, we identified CTF18-/- cells exhibiting strong sensitivity to Ara-C but not to cisplatin, which is a phenotypic trait observed in POLE1D269A/- cells [18]. We found that the loss of CTF18 sensitizes wild-type cells but not POLE1D269A/- cells to Ara-C. This epistatic relationship between CTF18-/- and POLE1D269A/- indicates that these two factors are closely related in the cellular response to Ara-C. Consistently, we found that CTF18-/-, POLE1D269A/-, and CTF18-/-/POLE1D269A/- cells showed comparable levels of reduction in replication fork speed after Ara-C treatment. Taken together, our data demonstrate that Polε exonuclease and CTF18 collaborate in the maintenance of the replication fork at the site of Ara-CMP incorporation.