The DNA replication machinery stalls at damaged sites on templates, but normally restarts by switching to a specialized DNA polymerase(s) that carries out translesion DNA synthesis (TLS). In human cells, DNA polymerase g (polg) accumulates at stalling sites as nuclear foci, and is involved in ultraviolet (UV)-induced TLS. Here we show that polg does not form nuclear foci in RAD18 À/À cells after UV irradiation. Both Rad18 and Rad6 are required for polg focus formation. In wild-type cells, UV irradiation induces relocalization of Rad18 in the nucleus, thereby stimulating colocalization with proliferating cell nuclear antigen (PCNA), and Rad18/Rad6-dependent PCNA monoubiquitination. Purified Rad18 and Rad6B monoubiquitinate PCNA in vitro. Rad18 associates with polg constitutively through domains on their C-terminal regions, and this complex accumulates at the foci after UV irradiation. Furthermore, polg interacts preferentially with monoubiquitinated PCNA, but pold does not. These results suggest that Rad18 is crucial for recruitment of polg to the damaged site through protein-protein interaction and PCNA monoubiquitination.

Translesion DNA synthesis (TLS) and homologous DNA recombination (HR) are two major postreplicational repair (PRR) pathways. The REV3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase ζ, which is involved in mutagenic TLS. To investigate the role of REV3 in vertebrates, we disruped the gene in chicken DT40 cells. REV3−/− cells are sensitive to various DNA‐damaging agents, including UV, methyl methanesulphonate (MMS), cisplatin and ionizing radiation (IR), consistent with its role in TLS. Interestingly, REV3−/− cells showed reduced gene targeting efficiencies and significant increase in the level of chromosomal breaks in the subsequent M phase after IR in the G2 phase, suggesting the involvement of Rev3 in HR‐mediated double‐strand break repair. REV3−/− cells showed significant increase in sister chromatid exchange events and chromosomal breaks even in the absence of exogenous genotoxic stress. Furthermore, double mutants of REV3 and RAD54, genes involved in HR, are synthetic lethal. In conclusion, Rev3 plays critical roles in PRR, which accounts for survival on naturally occurring endogenous as well as induced damages during replication.

We have investigated mechanisms that recruit the translesion synthesis (TLS) DNA polymerase Polκ to stalled replication forks. The DNA polymerase processivity factor PCNA is monoubiquitinated and interacts with Polκ in cells treated with the bulky adduct-forming genotoxin benzo[a]pyrene dihydrodiol epoxide (BPDE). A monoubiquitination-defective mutant form of PCNA fails to interact with Polκ. Small interfering RNA-mediated downregulation of the E3 ligase Rad18 inhibits BPDE-induced PCNA ubiquitination and association between PCNA and Polκ. Conversely, overexpressed Rad18 induces PCNA ubiquitination and association between PCNA and Polκ in a DNA damage-independent manner. Therefore, association of Polκ with PCNA is regulated by Rad18-mediated PCNA ubiquitination. Cells from Rad18 −/− transgenic mice show defective recovery from BPDE-induced S-phase checkpoints. In Rad18 −/− cells, BPDE induces elevated and persistent activation of checkpoint kinases, indicating persistently stalled forks due to defective TLS. Rad18-deficient cells show reduced viability after BPDE challenge compared with wild-type cells (but survival after hydroxyurea or ionizing radiation treatment is unaffected by Rad18 deficiency). Inhibition of RPA/ATR/Chk1-mediated S-phase checkpoint signaling partially inhibited BPDE-induced PCNA ubiquitination and prevented interactions between PCNA and Polκ. Taken together, our results indicate that ATR/Chk1 signaling is required for Rad18-mediated PCNA monoubiquitination. Recruitment of Polκ to ubiquitinated PCNA enables lesion bypass and eliminates stalled forks, thereby attenuating the S-phase checkpoint.

We initially performed exome-sequencing 11 of the two UV S S-A patients, Kps3 and XP24KO (details described in Methods, Supplementary Note, Table 2c). The patients were homozygous for c.367A>T mutation in UVSSA, which led to a premature termination, p.Lys123* (Fig. 1a, b). We identified the same homozygous mutation in Kps2 (sib. of Kps3), and a homozygous c.87delG, causing a frameshift p.Ile31Phefs*9, in an Israeli patient UV S S24TA (Fig. 1b, c, Supplementary Note, Supplementary Fig. 1). The identified mutations are summarized in Fig. 1d. We did not detect the 80kDa UVSSA protein in any of the UV S S-A patients (Fig. 1e). We additionally examined several mild xeroderma pigmentosum (XP) cases; in one such case, XP70TO 12 (Supplementary Table 1), we identified a homozygous p.Cys32Arg, in the UVSSA (Fig. 1c, d), implying that XP70TO is also in the UV S S-A group. The mutant protein was stably expressed in XP70TO cells (Fig. 1f, Supplementary Fig. 2a-d). 4Allele frequencies of the identified mutations in a control population were examined (Supplementary Note, Supplementary Fig. 3a). Haploinsufficiency for UVSSA is negligible as the parents of Kps2/Kps3 showed no symptoms 4 . In parallel with exome-sequencing, we performed whole-genome SNP-genotyping to identify runs-of-homozygosity (ROH) shared among the patients. We identified three overlapping-ROHs (> 1Mbps) on autosomes, one of which encompasses the UVSSA locus (Fig. 1g, Supplementary Table 3a, b, Supplementary Fig. 3b, c). No chromosome copy number variation was detected (Supplementary Fig. 3d).The above findings strongly suggest that the mutations in UVSSA in the UV S S-A patients are causal for the disease; we therefore, next examined the NER-activities in the UV S S-A cells (Fig. 2). Unscheduled-DNA-synthesis (UDS 13 , defective in XP) was nearly normal; however, RNA-synthesis-recovery (RRS 14 , defective in UV S S and in CS) was reduced in all cell-strains mutated in UVSSA ( Fig. 2a, b; UDS and RRS were measured using a recently-developed rapid non-radioactive system 15,16 ). Similarly, siRNA-based depletion of the UVSSA gene (Fig. 2c) caused a drastic reduction of RRS (Fig. 2d, Supplementary Fig. 4), whereas UDS was unaffected (Fig. 2e). Ectopic-expression of the wild-type UVSSA cDNA in UV S S-A cells restored normal RRS ( Fig. 2f; V5-tagged-UVSSA immunofluorescent-staining shown in Fig. 2g), while it did not affect RRS-level in normal, CS-A, or CS-B cells; neither ERCC8 nor ERCC6 cDNA expression in UV S S-A cells restored the RRS-level.We conclude that KIAA1530/UVSSA is the causal gene for UV S S-A.ERCC8 and ERCC6 genes are responsible for both CS and UV S S 7,8 . To evaluate whether UVSSA mutations may also result in CS-phenotypes, we sequenced 5 the UVSSA gene of 61 CS-patients whose genetic defects had not yet been determined (Supplementary Table 4). We found no obvious mutations except for four novel heterozygous changes. These changes as well as the SNPs, also found in control and UV S S-A individuals, do not affect the RRS-activity (Suppleme...

Postreplication repair functions in gap-filling of a daughter strand on replication of damaged DNA. The yeast Saccharomyces cerevisiae Rad18 protein plays a pivotal role in the process together with the Rad6 protein. Here, we have cloned a human homologue of RAD18, hRAD18. It maps on chromosome 3p24 -25, where deletions are often found in lung, breast, ovary, and testis cancers. In vivo, hRad18 protein binds to hHR6 protein through a conserved ring-finger motif. Stable transformants with hRad18 mutated in this motif become sensitive to UV, methyl methanesulfonate, and mitomycin C, and are defective in the replication of UV-damaged DNA. Thus, hRAD18 is a functional homologue of RAD18.