The main findings of this study are (i) Rad18 pathway activation occurs during G0, G1 and S-phase in response to both UV- and H2O2-induced DNA lesions in primary human cells; (ii) Rad18 prevents acquisition of DSB specifically after acquisition of oxidative DNA damage during G1; (iii) the role of Rad18 in preventing H2O2-induced DSB during G1 is non-essential owing to backup NHEJ-mediated DSB repair; (iv) in contrast with its redundant role in G1, Rad18 plays an essential role in facilitating completion of DNA replication and conferring cell survival after oxidative injury in S-phase. We conclude that Rad18 plays distinct roles in protecting the genome from oxidative DNA damage in different cell cycle stages.
The synergistic effect of Rad18- and NHEJ-ablation on H2
-sensitivity in G1
is unexpected. In previous studies (using asynchronous DT40 cells), loss of NHEJ rescued the DNA damage-sensitivity of Rad18-deficient cells (51
). The restoration of normal damage tolerance in Rad18−/−
DT40 cells after LigIV
-deletion was attributed to loss of NHEJ-mediated toxic end-joining events and redirecting of DNA damage processing to error-free homologous recombination (HR) during S-phase. Thus, our analysis of G1
-synchronized cells has revealed a new relationship between the Rad18 pathway and NHEJ.
As described in , our work indicates that H2O2-induced lesions acquired during G1 lead to Rad18-mediated PCNA mono-ubiquitination and Polη recruitment, facilitating repair synthesis at single-stranded gaps. In the absence of Rad18/Polη, SSB repair synthesis is compromised, leading to breaks in both strands. The resulting DSB activate ATM but are repaired via NHEJ conferring DNA damage tolerance.
Figure 8. Hypothetical model for functional redundancy of Rad18 and NHEJ in responding to oxidative DNA damage during G1. During G1, H2O2-treatment generates SSB. The exposed ssDNA is RPA-coated leading to Rad18 recruitment and PCNA mono-ubiquitinated by Rad18 (more ...)
-specificity of ATM activation by H2
in our experiments is also unexpected, as ATM is well known to mediate DSB-induced S-phase checkpoints (52
). It is possible that replication-coupled mechanisms for processing of oxidative lesions lead to S-phase-specific DNA structures that fail to activate ATM or are rapidly channelled through the HR pathway. It will be interesting to determine whether preventing HR during S-phase recapitulates the persistence of DSB and ATM hyper-phosphorylation phenotype we observed in H2
Our results complement and extend recent reports from Lehmann (24
) and Kannouche (25
) groups: Lehmann and colleagues (24
) demonstrated that nucleotide excision repair (NER) of UV-induced DNA damage in quiescent growth-arrested cells is Polκ-dependent. Kannouche and colleagues (25
) described TLS pathway activation in response to H2
in both growth-arrested (quiescent) and exponentially growing cells.
Our findings demonstrating DNA synthesis-independent PCNA mono-ubiquitination and Polη recruitment to chromatin are fully consistent with both studies. However, in contrast with the Lehmann and Kannouche groups (who compared G0
and asynchronous cells), we systematically defined responses to both H2
and UV in three cell cycle stages: G0
, and S-phase. Surprisingly, we show that defective TLS (achieved by Rad18 or Polη depletion) elicits a robust ATM response only outside of S-phase. Moreover, we show that the ATM pathway activation of TLS-defective cells occurs specifically in response to H2
(but not UV). Interestingly, however, the increased DSB formation in Rad18-deficient HDF after H2
treatment during G1
is not associated with increased lethality because of the high-DSB repair capacity in those cells. On the other hand, we show that Rad18-deficient cells are sensitive to H2
treatment during S-phase. Thus, the reported H2
-sensitivity of asynchronous Polη-deficient cells (25
) most likely resulted from post-replication repair defects during S-phase.
The RPA-dependence of PCNA mono-ubiquitination during G1
is fully consistent with the TLS activation mechanism proposed by Ulrich and colleagues (9
) involving Rad18 recruitment to RPA-coated ssDNA. It is important, therefore, to consider mechanisms by which RPA-coated DNA is generated outside of S-phase. NER incision events lead to ssDNA patches of ~30 bp. The minimal length of ssDNA that can bind RPA interaction site is eight nucleotides, and RPA has high affinity for oligonucleotides of 20–30 residues (54
). Therefore, NER of bulky lesions has the potential to generate the RPA-coated ssDNA intermediates required for Rad18 recruitment and PCNA mono-ubiquitination. Indeed, Ogi et al.
) demonstrated that NER-deficient xeroderma pigmentosum cells were defective for recruitment of TLS polymerases to chromatin in non-cycling cultures.
In contrast with the bulky UV lesions (that are processed by NER) H2
induces many forms of base damage, including 8-oxodG. Damaged bases, such as 8-oxodG, are repaired by short-patch and long-patch BER pathways that generate ssDNAs of 1 and 2–12 nucleotides, respectively (55
). Therefore, BER alone is unlikely to generate of sufficient ssDNA to trigger an RPA-mediated TLS response. In our experimental system, BER-deficiency resulting from knockdown of Ape1, or pharmacological inhibition of PARP did not affect PCNA mono-ubiquitination during G1
(Supplementary Figure S3
). The elegant study by Zlatanou et al.
) also found that BER is dispensable for PCNA mono-ubiquitination during G0
. Interestingly, however, those workers observed that the mismatch repair (MMR) proteins MSH2 and MSH6 are specifically required for PCNA mono-ubiquitination after H2
treatment. Therefore, Kannouche and colleagues suggest that clustered DNA damage resulting from H2
-induced oxidative lesions is recognized by MSH2–MSH6, promoting loading of Exo1 (or other exonucleases) that generate sufficient ssDNA for Rad18 recruitment, PCNA mono-ubiquitination and ensuing repair synthesis (56
). The MMR-mediated mechanism proposed by Kannouche and co-workers likely contributes to TLS-pathway activation in our experimental system.
In our study, H2
induced substantial (~50–70-fold) increases in loading of PCNA onto chromatin after H2
treatments during G0
(but not during S-phase when PCNA is already largely chromatin bound). It will be interesting to identity of the clamp loader(s) involved in responses to H2
-induced damage. MSH2 is reported to interact with PCNA (57
). It is possible that MMR proteins contribute to both PCNA loading (via direct interactions) and PCNA mono-ubiquitination (by generating ssDNA gaps necessary for RPA loading and Rad18 recruitment).
As Rad18 and TLS polymerases facilitate repair of both UV- and H2
-induced discontinuities in dsDNA (24
), it is interesting that the ATM ‘hyper-phosphorylation’ phenotype of TLS-deficient G1
cells is highly specific to H2
Paull and colleagues (58
) have demonstrated that ATM may be activated directly by oxidative stress. However, because depletion of Rad18 or of Polη promotes ATM S1981 phosphorylation by H2
, we consider it unlikely that the increased ATM phosphorylation associated with TLS-deficiency is related to direct effects of reactive oxygen species on ATM activity. Moreover, Walker and colleagues (60
) have shown that the redox-dependent activation of ATM occurs in the cytosol. All the ATM immunoblots presented here were from chromatin fractions. We have never detected S1981 phosphorylation of cytosolic ATM, also arguing against a mechanism involving direct activation of ATM by H2
under our experimental conditions.
Another possible explanation for the H2
-specificity of ATM activation in TLS-deficient cells is that Polη is redundant with other DNA polymerases for repair synthesis at UV-induced discontinuities but not at H2
-induced SSB. The differential ATM activation in response to H2
and UV could also be explained by the nature of the single-stranded breaks induced by the two agents. For example, oxidative damage may be clustered in H2
-treated cells, whereas it is unlikely that UV fluences used in our experiments result in bi-stranded or tandem clusters of CPD. It is also possible that the putative MMR-dependent exonuclease(s) activated during processing of oxidative lesions (25
) generates longer patches of RPA–ssDNA (relative to the ~30 bp patches generated during NER of UV lesions) that are more prone to breakage if left unrepaired.
Rad18 is implicated in multiple DNA repair pathways, yet Rad18-
deficient mice display only mild phenotypes (61
). Therefore, redundant mechanisms likely exist to maintain the genome and confer DNA damage tolerance in the absence of Rad18. We show here that H2
-induced DSB generated in Rad18/Polη-deficient cells can be repaired by NHEJ. Therefore, it is possible that combined defects in Rad18 and other DNA repair genes (including DNA-PK or other NHEJ genes) will reveal more profound DNA damage tolerance phenotypes than are evident in Rad18
mutant mice. The finding that the Rad18 pathway is activated by H2
in a DNA replication-independent manner suggests a potential role for Rad18 in tolerance of oxidative stress-induced DNA damage in non-proliferating cells, such as neurons and cardiomyocytes, that can experience considerable ROS exposure in vivo
. Further experiments are underway to test roles of Rad18 in tolerance of ROS-induced DNA lesions in non-dividing cells in vivo