In this study, we present novel insight into the molecular mechanism of UV-induced H2A ubiquitination. Our data show that after UV damage, the Ub E3 ligase RNF8, in concert with the E2-conjugating enzyme Ubc13, is essential for the NER-dependent Ub modification of histone H2A. In a previous study, we reported that UV-induced ubiquitination of histone H2A required Ring2 (Bergink et al., 2006
). Notably, Ring2 knockdown also results in a severe reduction of basal H2A ubiquitination as well as the nuclear Ub pool. Thus, Ring2 knockdown is likely to disturb the cellular Ub equilibrium (Groothuis et al., 2006
) and may affect downstream-specific ubiquitination reactions such as the RNF8-dependent H2A ubiquitination. In contrast, RNF8 knockdown inhibits only UV-induced and not basal H2A ubiquitination (; Huen et al., 2007
; Mailand et al., 2007
). Furthermore, we show that RNF8 is recruited to the damaged DNA, suggesting that RNF8 is the DNA damage–specific H2A E3 ligase.
Intriguingly, a significant proportion of Ubc13 was found to accumulate at the site of DNA damage, indicating that a large part of the pool of Ubc13 proteins plays a role during UV-induced DNA repair. Recently, it was also observed that a large part of the total amount of Ubc13 is involved during the DSB-induced DDR, as was shown by the accumulation of Ubc13 at laser-induced DSB damage (Ikura et al., 2007
). This indicates that Ubc13 plays an important role during DNA repair in addition to its involvement in many other biological processes (Pickart, 2000
). Because RNF8 recruitment to damaged DNA is independent on Ubc13 (unpublished data), we assume that Ubc13 depends on RNF8 recruitment, which is in line with the described interaction of Ubc13 with the RING finger domain of RNF8 (Plans et al., 2006
). The strong accumulation of Ubc13 at the site of damage, which is surprising for such a usually less-specific functioning, E2-conjugating enzyme (in contrast to the more substrate-specific E3 ligases), indicates its important role during UV-induced DDR. Ubc13 is, for the majority, not involved in the core NER machinery because Ubc13 depletion has no detectable effect on UDS () or XPA accumulation (Fig. S1 d). Most likely, Ubc13 plays a role in the downstream UV-induced DDR together with RNF8.
Our data () show that the molecular mechanism that induces ubiquitination of H2A after genotoxic stress, including the recruitment of downstream factors 53BP1 and BRCA1, is highly conserved between DSB- and UV-induced DDR. This is remarkable considering the important mechanistic differences between DSB and UV damage repair, which may explain the variation in observed timing. MDC1 and RNF8 recruitment are early events in the DSB-induced DDR (Mailand et al., 2007
), although it is a relatively late event after UV-induced DNA damage. It is possible that this difference is caused by a much faster processing of DSB breaks, resulting in a fast ATM activation compared with a slower ATR activation by UV lesion processing.
Importantly, we found factors (e.g., MDC1, 53BP1, and Brca1) to be recruited to UV-damaged DNA that were previously known only to play a role in the DSB response. Until now, their involvement in UV–DDR was unanticipated, although recently, some were found to be phosphorylated by ATR after UV damage, such as MDC1 (Stewart et al., 2003
), RAP80 (Yan et al., 2008
), and 53BP1 (Jowsey et al., 2007
). Although these proteins are maximally phosphorylated within minutes upon IR via ATM, after UV, their phosphorylation becomes apparent after 60 min and keeps increasing in time, showing that the UV–DDR response via this RNF8 pathway has indeed slower kinetics.
The question of why these DSB–DDR factors (including MDC1, 53BP1, and Brca1) are also recruited to chromatin after UV damage is more difficult to answer. Genetic insults may ring a “general alarm bell,” resulting in the assembly of a toolbox of diverse DDR proteins near the lesion irrespective of the type of DNA damage. This enables the cell to facilitate different response pathways, but only a specific subset will be used if needed (Harper and Elledge, 2007
). This might also explain why RNF8 is observed to accumulate at stalled replication forks (Sakasai and Tibbetts, 2008
). The recruitment of the DDR proteins such as MDC1, 53BP1, and BRCA1 may represent an extra line of defense against the UV-induced damage. Normally, only the NER-specialized DNA repair enzymes will be used. However, in the rare event that a DSB originates from a UV lesion during replication, the DSB-involved proteins that were present “just in case” at the damaged chromatin can swiftly be used.
Alternatively, the same pathways, and thus the same proteins, are used as cells use similar crisis management strategies triggered by different repair machineries. H2A ubiquitination increases the local concentration of 53BP1 and BRCA1 that enhances activation of the cell cycle checkpoint kinases Chk1 and Chk2 (Stucki and Jackson, 2006
). siRNA-mediated down-regulation of MDC1 results in a lower UV-induced Chk1 phosphorylation, indicating that this pathway is involved in the checkpoint induction and maintenance (). Chk1 and Chk2 play a crucial role in DDR by, for example, regulating the S to G2 and G2/M checkpoints (Niida and Nakanishi, 2006
). Both checkpoints are implicated in the cellular survival of IR and UV, which may explain the use of a common DDR pathway.