RAD18 promotes DNA double-strand break repair during G1 phase through chromatin retention of 53BP1.

Recruitment of RAD18 to stalled replication forks facilitates monoubiquitination of PCNA during S-phase, promoting translesion synthesis at sites of UV irradiation-induced DNA damage. In this study, we show that RAD18 is also recruited to ionizing radiation (IR)-induced sites of DNA double-strand breaks (DSBs) forming foci which are co-localized with 53BP1, NBS1, phosphorylated ATM, BRCA1 and gamma-H2AX. RAD18 associates with 53BP1 and is recruited to DSB sites in a 53BP1-dependent manner specifically during G1-phase, RAD18 monoubiquitinates KBD domain of 53BP1 at lysine 1268 in vitro. A monoubiquitination-resistant 53BP1 mutant harboring a substitution at lysine 1268 is not retained efficiently at the chromatin in the vicinity of DSBs. In Rad18-null cells, retention of 53BP1 foci, efficiency of DSB repair and post-irradiation viability are impaired compared with wild-type cells. Taken together, these results suggest that RAD18 promotes 53BP1-directed DSB repair by enhancing retention of 53BP1, possibly through an interaction between RAD18 and 53BP1 and the modification of 53BP1.

A new disorder in UV-induced skin cancer with defective DNA repair distinct from xeroderma pigmentosum or Cockayne syndrome.

We report the characterization of a Japanese woman who exhibited many freckles and skin cancers in sun-exposed areas, but displayed no photosensitivity. Fibroblasts (KPSX7) derived from this patient showed similar UV sensitivity to that of normal human fibroblasts. The KPSX7 cells showed normal levels of unscheduled DNA synthesis, recovery of RNA synthesis, recovery of replicative DNA synthesis, protein-binding ability to UV-damaged DNA, and post-translational modification of xeroderma pigmentosum (XP) C. These results indicate that the patient had neither XP nor Cockayne syndrome. Although these results suggest that the KPSX7 cells were proficient in nucleotide excision repair activity, host-cell reactivation (HCR) activity of KPSX7 cells was reduced. Furthermore, introduction of UV damage endonuclease into the cells restored repair activity in the HCR assay to almost normal levels. These results indicate that KPSX7 cells are defective for some types of repair activity in UV-damaged DNA. In summary, the patient had a previously unknown disorder related to UV-induced carcinogenesis, with defective DNA repair.

Human RAD18 is involved in S phase-specific single-strand break repair without PCNA monoubiquitination.

Switching from a replicative to a translesion polymerase is an important step to further continue on replication at the site of DNA lesion. Recently, RAD18 (a ubiquitin ligase) was shown to monoubiquitinate proliferating cell nuclear antigen (PCNA) in cooperation with RAD6 (a ubiquitin-conjugating enzyme) at the replication-stalled sites, causing the polymerase switch. Analyzing RAD18-knockout (RAD18-/-) cells generated from human HCT116 cells, in addition to the polymerase switch, we found a new function of RAD18 for S phase-specific DNA single-strand break repair (SSBR). Unlike the case with polymerase switching, PCNA monoubiquitination was not necessary for the SSBR. When compared with wild-type HCT116 cells, RAD18-/- cells, defective in the repair of X-ray-induced chromosomal aberrations, were significantly hypersensitive to X-ray-irradiation and also to the topoisomerase I inhibitor camptothecin (CPT) capable of inducing single-strand breaks but were not so sensitive to the topoisomerase II inhibitor etoposide capable of inducing double-strand breaks. However, such hypersensitivity to CPT observed with RAD18-/- cells was limited to only the S phase due to the absence of the RAD18 S phase-specific function. Furthermore, the defective SSBR observed in S phase of RAD18-/- cells was also demonstrated by alkaline comet assay.

A human DNA polymerase eta complex containing Rad18, Rad6 and Rev1; proteomic analysis and targeting of the complex to the chromatin-bound fraction of cells undergoing replication fork arrest.

DNA polymerase eta (Poleta) is responsible for efficient translesion synthesis (TLS) past cis-syn cyclobutane thymine dimers (TT dimers), the major DNA lesions induced by UV irradiation. Loss of human Poleta leads to xeroderma pigmentosum variant syndrome, clearly indicating that Poleta plays a vital role in preventing skin cancer caused by exposure to sunlight. To further examine Poleta functions and the mechanisms that regulate this important protein, Poleta complexes were purified from HeLa cells over-expressing epitope-tagged Poleta, and polypeptides associated with Poleta, including Rad18, Rad6 and Rev1, were identified by a combination of mass spectrometry and Western blot analysis. The chromatin-bound fractions of cells subjected to UV irradiation, S phase synchronization, or S phase arrest were specifically enriched in such complexes. These results suggest that arrested replication forks strengthen interactions among Poleta, Rad18/Rad6 and Rev1, consistent with the requirement for effective TLS by Poleta at sites of DNA lesions.

Effect of DNA repair protein Rad18 on viral infection.

Host factors belonging to the DNA repair machineries are assumed to aid retroviruses in the obligatory step of integration. Here we describe the effect of DNA repair molecule Rad18, a component of the post-replication repair pathway, on viral infection. Contrary to our expectations, cells lacking Rad18 were consistently more permissive to viral transduction as compared to Rad18(+/+) controls. Remarkably, such susceptibility was integration independent, since retroviruses devoid of integration activity also showed enhancement of the initial steps of infection. Moreover, the elevated sensitivity of the Rad18(-/-) cells was also observed with adenovirus. These data indicate that Rad18 suppresses viral infection in a non-specific fashion, probably by targeting incoming DNA. Furthermore, considering data published recently, it appears that the interactions between DNA repair components with incoming viruses, often result in inhibition of the infection rather than cooperation toward its establishment.

Rad18 regulates DNA polymerase kappa and is required for recovery from S-phase checkpoint-mediated arrest.

We have investigated mechanisms that recruit the translesion synthesis (TLS) DNA polymerase Polkappa to stalled replication forks. The DNA polymerase processivity factor PCNA is monoubiquitinated and interacts with Polkappa 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 Polkappa. Small interfering RNA-mediated downregulation of the E3 ligase Rad18 inhibits BPDE-induced PCNA ubiquitination and association between PCNA and Polkappa. Conversely, overexpressed Rad18 induces PCNA ubiquitination and association between PCNA and Polkappa in a DNA damage-independent manner. Therefore, association of Polkappa 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 Polkappa. Taken together, our results indicate that ATR/Chk1 signaling is required for Rad18-mediated PCNA monoubiquitination. Recruitment of Polkappa to ubiquitinated PCNA enables lesion bypass and eliminates stalled forks, thereby attenuating the S-phase checkpoint.

Differential regulation of Rad18 through Rad6-dependent mono- and polyubiquitination.

Rad18 is involved in postreplication repair mainly through monoubiquitination of proliferating cell nuclear antigen (PCNA). Here we show that Rad18 protein was detected in human cells as two major bands at 75 and 85 kDa by Western blot. The bands were identified as nonubiquitinated and monoubiquitinated forms of Rad18, respectively, by mass spectrometry. Multiple ubiquitinated bands of Rad18 were detected in vitro in the presence of E1, E2 (Rad6), and methylated ubiquitin, indicating that Rad18 was monoubiquitinated at multiple sites through autoubiquitination. Rad18 self-associates, and this interaction was abolished by replacing one of the conserved cysteine residues with phenylalanine in the zinc finger domain (C207F). In the C207F mutant Rad18, monoubiquitination of Rad18 was not observed in vivo, suggesting that self-association was critical for monoubiquitination. Monoubiquitinated Rad18 was detected mainly in the cytoplasm, whereas nonubiquitinated Rad18 was detected predominantly in the nuclei. Furthermore, Rad18 was shown to be polyubiquitinated in cells treated with proteasome inhibitors. Purified Rad18 was also polyubiquitinated in an in vitro system containing E1, E2 (Rad6), and ubiquitin, and it was degraded by the addition of proteasomes. These results suggest that the amount of Rad18 in the nucleus is regulated differentially by mono- and polyubiquitination.

Roles of MGMT and MLH1 proteins in alkylation-induced apoptosis and mutagenesis.

To examine involvement of mismatch repair system in alkylation-induced apoptosis and mutagenesis, cell lines defective in the Mgmt gene encoding a DNA repair enzyme, O(6)-methylguanine-DNA methyltransferase, and/or the Mlh1 gene encoding a protein involved in mismatch repair were established from gene-targeted mice. Mgmt(-/-) cells are hypersensitive to the killing effect of N-methyl-N-nitrosourea (MNU) and this effect of MNU was overcome by introducing an additional mutation in the Mlh1 gene. Mgmt(-/-)Mlh1(-/-) cells are more resistant to MNU than are wild-type cells. When the human Mgmt cDNA sequence with a strong promoter was introduced, the wild-type cells acquired the same high level of resistance to MNU as that of Mgmt(-/-)Mlh1(-/-) cells. Although no apparent increase in MNU-induced mutant frequency was observed in such methyltransferase-overproducing wild-type cells, mutant frequency of Mgmt(-/-)Mlh1(-/-) cells became 10-fold higher after being treated with MNU. Mgmt(-/-)Mlh1(+/-) cells carrying approximately half the normal level of MLH1 protein showed a normal level of spontaneous mutant frequency, yet were still highly responsive to the mutagenic effect of the alkylating carcinogen. This haploinsufficient character of Mlh1 mutation was also observed in cell survival assays; Mgmt(-/-)Mlh1(+/-) cells were as resistant to MNU as were Mgmt(-/-)Mlh1(-/-) cells. While caspase-3 was induced in Mgmt(-/-)Mlh1(+/+) cells after treatment with MNU, no induction occurred in Mgmt(-/-)Mlh1(+/-) cells or in Mgmt(-/-)Mlh1(-/-) cells. The cellular content of MLH1 protein seems to be critical for determining if damaged cells enter into either a death or mutation-inducing pathway. The haploinsufficient phenotype of Mlh1-heterozygous cells may be explained by competition in heterodimer formation between MLH1 homologues.

Ultraviolet-sensitive syndrome cells are defective in transcription-coupled repair of cyclobutane pyrimidine dimers.

Patients with ultraviolet-sensitive syndrome (UV(S)S) are sensitive to sunlight, but present neither developmental nor neurological deficiencies. Complementation studies with hereditary DNA repair syndromes show that UV(S)S is distinct from all known xeroderma pigmentosum (XP) and Cockayne syndrome (CS) groups. UV(S)S cells exhibit some characteristics typical of CS, including normal global genomic (GGR) repair of UV-photoproducts, poor clonal survival and defective recovery of RNA synthesis after UV exposure. Those observations have led us to suggest that UV(S)S cells, like those from CS, are defective in transcription-coupled repair (TCR) of cyclobutane pyrimidine dimers (CPD). We have now examined the repair of CPD in the transcribed and non-transcribed strands of the active dihydrofolate reductase (DHFR) and p53 genes, and of the silent alpha-fetoprotein (AFP) and mid-size neurofilament (NF-M) genes in normal human cells and in cells belonging to UV(S)S and CS complementation group B. Our results provide compelling evidence that the UV(S)S gene is essential for TCR of CPD and probably other bulky DNA lesions. As a possible distinction between UV(S)S and CS patients, we postulate that the UV(S)S gene may not be required for TCR of oxidative lesions. We have also found that repair of CPD in either DNA strand of the genomic fragments examined, occurs at a slower rate in TCR-deficient cells than in the non-transcribed strands in normal cells; we suggest that in the absence of TCR, global repair complexes have hindered access to lesions in genomic regions that extend beyond individual transcription units.