RAD18- and BRCA1-dependent pathways promote cellular tolerance to the nucleoside analog ganciclovir.

Ganciclovir (GCV) is a clinically important drug as it is used to treat viral infections. GCV is incorporated into the DNA during replication, where it interferes with subsequent replication on GCV-incorporated templates. However, the effects of GCV on the host genome and the mechanisms underlying cellular tolerance to GCV remain unclear. In this study, we explored these mechanisms using a collection of mutant DT40 cells. We identified RAD17/-, BRCA1-/-, and RAD18-/- cells as highly GCV-sensitive. RAD17, a component of the alternative checkpoint-clamp loader RAD17-RFC, was required for the activation of the intra-S checkpoint following GCV treatment. BRCA1, a critical factor for promoting homologous recombination (HR), was required for suppressing DNA double-strand breaks (DSBs). Moreover, RAD18, an E3-ligase involved in DNA repair, was critical in suppressing the aberrant ligation of broken chromosomes caused by GCV. We found that BRCA1 suppresses DSBs through HR-mediated repair and template switching (TS)-mediated damage bypass. Moreover, the strong GCV sensitivity of BRCA1-/- cells was rescued by the loss of 53BP1, despite the only partial restoration in the sister chromatid exchange events which are hallmarks of HR. These results indicate that BRCA1 promotes cellular tolerance to GCV through two mechanisms, TS and HR-mediated repair.

Dominant roles of BRCA1 in cellular tolerance to a chain-terminating nucleoside analog, alovudine.

Alovudine is a chain-terminating nucleoside analog (CTNA) that is frequently used as an antiviral and anticancer agent. Generally, CTNAs inhibit DNA replication after their incorporation into nascent DNA during DNA synthesis by suppressing subsequent polymerization, which restricts the proliferation of viruses and cancer cells. Alovudine is a thymidine analog used as an antiviral drug. However, the mechanisms underlying the removal of alovudine and DNA damage tolerance pathways involved in cellular resistance to alovudine remain unclear. Here, we explored the DNA damage tolerance pathways responsible for cellular tolerance to alovudine and found that BRCA1-deficient cells exhibited the highest sensitivity to alovudine. Moreover, alovudine interfered with DNA replication in two distinct mechanisms: first: alovudine incorporated at the end of nascent DNA interfered with subsequent DNA synthesis; second: DNA replication stalled on the alovudine-incorporated template strand. Additionally, BRCA1 facilitated the removal of the incorporated alovudine from nascent DNA, and BRCA1-mediated homologous recombination (HR) contributed to the progressive replication on the alovudine-incorporated template. Thus, we have elucidated the previously unappreciated mechanism of alovudine-mediated inhibition of DNA replication and the role of BRCA1 in cellular tolerance to alovudine.

The proofreading exonuclease of leading-strand DNA polymerase epsilon prevents replication fork collapse at broken template strands.

Leading-strand DNA replication by polymerase epsilon (Polϵ) across single-strand breaks (SSBs) causes single-ended double-strand breaks (seDSBs), which are repaired via homology-directed repair (HDR) and suppressed by fork reversal (FR). Although previous studies identified many molecules required for hydroxyurea-induced FR, FR at seDSBs is poorly understood. Here, we identified molecules that specifically mediate FR at seDSBs. Because FR at seDSBs requires poly(ADP ribose)polymerase 1 (PARP1), we hypothesized that seDSB/FR-associated molecules would increase tolerance to camptothecin (CPT) but not the PARP inhibitor olaparib, even though both anti-cancer agents generate seDSBs. Indeed, we uncovered that Polϵ exonuclease and CTF18, a Polϵ cofactor, increased tolerance to CPT but not olaparib. To explore potential functional interactions between Polϵ exonuclease, CTF18, and PARP1, we created exonuclease-deficient POLE1exo-/-, CTF18-/-, PARP1-/-, CTF18-/-/POLE1exo-/-, PARP1-/-/POLE1exo-/-, and CTF18-/-/PARP1-/- cells. Epistasis analysis indicated that Polϵ exonuclease and CTF18 were interdependent and required PARP1 for CPT tolerance. Remarkably, POLE1exo-/- and HDR-deficient BRCA1-/- cells exhibited similar CPT sensitivity. Moreover, combining POLE1exo-/- with BRCA1-/- mutations synergistically increased CPT sensitivity. In conclusion, the newly identified PARP1-CTF18-Polϵ exonuclease axis and HDR act independently to prevent fork collapse at seDSBs. Olaparib inhibits this axis, explaining the pronounced cytotoxic effects of olaparib on HDR-deficient cells.

CTF18-RFC contributes to cellular tolerance against chain-terminating nucleoside analogs (CTNAs) in cooperation with proofreading exonuclease activity of DNA polymerase ε.

Chemotherapeutic nucleoside analogs, such as cytarabine (Ara-C), are incorporated into genomic DNA during replication. Incorporated Ara-CMP (Ara-cytidine monophosphate) serves as a chain terminator and inhibits DNA synthesis by replicative polymerase epsilon (Polε). The proofreading exonuclease activity of Polε removes the misincorporated Ara-CMP, thereby contributing to the cellular tolerance to Ara-C. Purified Polε performs proofreading, and it is generally believed that proofreading in vivo does not need additional factors. In this study, we demonstrated that the proofreading by Polε in vivo requires CTF18, a component of the leading-strand replisome. We found that loss of CTF18 in chicken DT40 cells and human TK6 cells results in hypersensitivity to Ara-C, indicating the conserved function of CTF18 in the cellular tolerance of Ara-C. Strikingly, we found that proofreading-deficient POLE1D269A/-, CTF18-/-, and POLE1D269A/-/CTF18-/- cells showed indistinguishable phenotypes, including the extent of hypersensitivity to Ara-C and decreased replication rate with Ara-C. This observed epistatic relationship between POLE1D269A/- and CTF18-/- suggests that they are interdependent in removing mis-incorporated Ara-CMP from the 3' end of primers. Mechanistically, we found that CTF18-/- cells have reduced levels of chromatin-bound Polε upon Ara-C treatment, suggesting that CTF18 contributes to the tethering of Polε on fork at the stalled end and thereby facilitating the removal of inserted Ara-C. Collectively, these data reveal the previously unappreciated role of CTF18 in Polε-exonuclease-mediated maintenance of the replication fork upon Ara-C incorporation.

Chromatin determinants of the inner-centromere rely on replication factors with functions that impart cohesion.

Replication fork-associated factors promote genome integrity and protect against cancer. Mutations in the DDX11 helicase and the ESCO2 acetyltransferase also cause related developmental disorders classified as cohesinopathies. Here we generated vertebrate model cell lines of these disorders and cohesinopathies-related genes. We found that vertebrate DDX11 and Tim-Tipin are individually needed to compensate for ESCO2 loss in chromosome segregation, with DDX11 also playing complementary roles with ESCO2 in centromeric cohesion. Our study reveals that overt centromeric cohesion loss does not necessarily precede chromosome missegregation, while both these problems correlate with, and possibly originate from, inner-centromere defects involving reduced phosphorylation of histone H3T3 (pH3T3) in the region. Interestingly, the mitotic pH3T3 mark was defective in all analyzed replication-related mutants with functions in cohesion. The results pinpoint mitotic pH3T3 as a postreplicative chromatin mark that is sensitive to replication stress and conducts with different kinetics to robust centromeric cohesion and correct chromosome segregation.