Mitotic DNA synthesis in response to replication stress requires the sequential action of DNA polymerases zeta and delta in human cells

Abstract

Oncogene activation creates DNA replication stress (RS) in cancer cells, which can generate under-replicated DNA regions (UDRs) that persist until cells enter mitosis. UDRs also have the potential to generate DNA bridges in anaphase cells or micronuclei in the daughter cells, which could promote genomic instability. To suppress such damaging changes to the genome, human cells have developed a strategy to conduct ‘unscheduled’ DNA synthesis in mitosis (termed MiDAS) that serves to rescue under-replicated loci. Previous studies have shown that MiDAS proceeds via a POLD3-dependent pathway that shows some features of break-induced replication. Here, we define how human cells utilize both DNA gap filling (REV1 and Pol ζ) and replicative (Pol δ) DNA polymerases to complete genome duplication following a perturbed S-phase. We present evidence for the existence of a polymerase-switch during MiDAS that is required for new DNA synthesis at UDRs. Moreover, we reveal that, upon oncogene activation, cancer cell survival is significantly compromised when REV1 is depleted, suggesting that REV1 inhibition might be a feasible approach for the treatment of some human cancers.

FANCD2-dependent mitotic DNA synthesis relies on PCNA K164 ubiquitination

“…Previous studies investigating the role of PCNA ubiquitination in human cells relied on ectopic expression of PCNA K164R or PCNA-ubiquitin fusions in cells expressing wild-type PCNA, with or without knockdown of endogenous PCNA. 10 , 11 , 18 To understand the importance of PCNA K164 modification when stably expressed from the endogenous locus, we utilized CRISPR-Cas9-mediated gene targeting to knock in an A>G mutation in the codon for K164 of PCNA in RPE-1 cells ( Figure S1A ). PCR analyses identified mono- and biallelic targeting of PCNA ( Figures S1B and S1C ), and sequencing confirmed one homozygous PCNA K164R (2B10) and two hemizygous PCNA K164R/− (A1 and B1) clonal cell lines.…”

“…An increase in under-replicated genomic regions can lead to the formation of FANCD2 foci during, and activation of MiDAS as the cells’ final attempt to complete DNA replication prior to, cell division. 16 , 18 Whereas transformed cells utilize RAD52 and FANCD2 for MiDAS, non-transformed cell lines do not require RAD52 and are dependent on FANCD2. 16 To visualize APH-induced MiDAS, we pulse labeled cells with EdU and quantified the number of EdU and FANCD2 foci in nuclei that stained positive for histone H3 phosphorylation at serine 10 (pH3-S10), a hallmark of mitotic chromosomes ( Figure S5A ).…”

“… 16 , 17 Interestingly, a recent study found that PCNA K164 ubiquitination by RAD18 is required for MiDAS in cancer cell lines but did not investigate its necessity in non-transformed human cells. 18 …”

Transient Zn2+deficiency induces replication stress and compromises daughter cell proliferation

“…The distribution of fates shifts toward the CDK2 low population the longer a cell is in Zn 2+ deficient media (Figure 4E-F). Of note, the transmission of replication stress through mitosis is not accompanied by mitotic DNA synthesis (MiDAS) (Figure S7), which marks the transmission of unresolved replication stress to daughter cell quiescence (38, 39). These data demonstrate that cells pulsed with TPA during S-phase show a steady decrease in proliferative cells after division and reach a nadir during the middle of S-phase.…”

PARP2 promotes Break Induced Replication-mediated telomere fragility in response to replication stress

“…Pol z, along with the scaffolding factor REV1, can operate in G2 to promote replication of UV-damaged DNA 67 . Based on these observations, a very recent study has demonstrated that both Pol z and Pol d are required to promote MiDAS in a sequential manner 68 . More specifically, Pol z seems to act upstream of Pol d, which is recruited through an interaction of the REV1 interacting region (RIR) of POLD3 with REV1 and via a polymerase switch mechanism that remains unclear.…”