In this article, we have shown that deletion of the RSC2
gene in yeast or depletion of the orthologous BAF180 in human cells results in a pronounced decrease in the ubiquitination of PCNA following DNA damage or HU treatment relative to undepleted controls. In human cells, we have been able to attribute this reduction to a decreased concentration of the E3 ligase Rad18 in chromatin and remarkably to a decrease in chromatin-associated unmodified PCNA as well. All of these reductions are ~3- to 5-fold. None of these effects results from distortions of the cell cycle in the absence of BAF180. We can draw two major conclusions from our findings. First, BAF180 and by implication the PBAF complex, plays an important role in loading PCNA onto chromatin and enhancing its ubiquitination following DNA damaging treatment. It is of interest to note that two recent papers report on the role of other chromatin modifications/chromatin proteins on recruitment of PCNA to chromatin in human cells. Mono-methylation of histone H3 on lysine 56 and the HMGN1 protein are both important for recruitment of PCNA to chromatin to facilitate DNA replication (47
). Our second conclusion is that, whereas the reduced levels of unmodified and ubiquitinated PCNA result in a modest effect on fork progression, they have no effect on UV survival in human cells.
We previously showed that replacement of PCNA in MRC5 cells with mutant PCNA-K164R that cannot be ubiquitinated, resulted in a substantial reduction in UV survival (46
). Our current data suggest that, following DNA damage, cells can quite happily dispense with 70–80% of both unmodified and ubiquitinated PCNA with no significant effect on cell survival. (It should be noted that the absence of polη in XP variant cells confers only minor sensitivity to UV-induced cell killing, despite polη being a central player in TLS.) Gong et al
) found that a human cell line lacking the Swi–Snf component Brg1 was sensitive to UV-irradiation. However, there are two Swi–Snf complexes in human cells with several components in common, including Brg1. Hence, lack of Brg1 would result in both complexes being defective. In our work, we depleted BAF180, which is a specific component of only one of the complexes, leaving the other one intact. Our data are therefore not directly comparable with those of Gong et al.
A current model for replication at sites of DNA damage is that the PCNA-associated replication machinery is blocked at the site of damage, but the replicative helicase continues to unwind ahead of the fork, exposing single-stranded DNA at the fork. Two events then occur. One of these is the repriming of synthesis downstream of the lesion, using a new PCNA trimer. The other event is the recruitment of Rad18 by the single-stranded DNA to ubiquitinate PCNA. This in turn recruits a TLS polymerase to bypass the damage. Let us first consider the situation in which repriming occurs before TLS (B, mode 1). The reprimed replication machinery will be blocked at the next lesion and a further repriming event will take place. Each repriming event requires a new PCNA molecule and each blockage at a lesion results in a new ubiquitination event. If repriming occurs in this way, TLS will take place behind the fork. In an alternative mode (B, mode 2), TLS occurs at the fork, the blocked fork is then released to continue with the same PCNA trimer, and repriming may not be necessary. Since this PCNA is already ubiquitinated, another ubiquitination event is not required at the next damaged site encountered. Note that replicative polymerases operate equally well irrespective of whether PCNA is ubiquitinated or not (50
), and we have previously shown that PCNA remains ubiquitinated for many hours after UV-irradiation (46
In this scenario, we propose that PBAF is required to mobilize nucleosomes to allow repriming to take place. This involvement may be either to assist the helicase in unwinding the DNA ahead of the fork or to help the repriming event itself. The latter is perhaps less likely, as repriming takes place on single-stranded DNA, which might be expected to be free of nucleosomes. Several reports have shown that UV-blocked replication forks can restart efficiently by repriming (mode 1) under normal circumstances (51–53
). In the absence of PBAF, we suggest that there is a shift in the balance from mode 1 to mode 2, which results in reduced PCNA loading, less recruitment of Rad18 and decreased ubiquitination of PCNA. There is no intrinsic requirement for the involvement of more polη molecules in either mode. If the time interval between repriming and TLS in mode 1 is relatively short, the effect on fork progression need not be very dramatic, and there would not necessarily be any deleterious effect of shifting the balance from mode 1 to mode 2. This would correlate with our finding of a modest decrease in fork progression rate. The model is most easily applicable to events on the leading strand. We note however, that the decrease in chromatin-associated PCNA and ubiquitinated PCNA is >50% when PBAF is depleted, implying a role for PBAF in events on the lagging strand as well. The exact nature of this role must await further experimentation. Our model is highly speculative, but it does provide a satisfactory explanation for our otherwise apparently self-contradictory findings. It posits a role for PBAF/RSC in repriming beyond stalled forks, and predicts that the chromatin remodeller is located at the site of replication. This is consistent with our ChIP data showing that Rsc2 is indeed associated with chromatin in the vicinity of replication forks in yeast cells. In an elegant recent study, Cohen et al
) showed that BRG1, a core component of Swi–Snf complexes in mammalian cells, is localized at sites of DNA replication on chromatin fibres and is required for efficient fork progression. This work does not indicate which of the two mammalian Brg1-containing complexes, only one of which (PBAF) contains BAF180, is located and required at the replication forks. We did not find any effect of depleting BAF180 on fork progression in unirradiated cells. However, their results are consistent with our finding that Rsc2 is associated with the replication fork in yeast.
Our data demonstrate that RSC/PBAF has a major effect on PCNA ubiquitination in response to a range of DNA lesions, and that the complex is localized to sites of DNA replication in vivo
. We also find, consistent with previous reports, that INO80 facilitates PCNA ubiquitination (55
) and is localized to sites of DNA replication. INO80 contributes to PCNA ubiquitination only after MMS-induced DNA damage, but these results still raise the possibility that there are redundant functions of the two chromatin remodellers at stalled replication forks. However, RSC promotes nucleosome repositioning in vivo
), whereas INO80 removes H2A.Z/H2B dimers from nucleosomes and replaces them with H2A/H2B dimers (37
). This difference in remodelling activity strongly suggests that the two complexes will promote PCNA ubiquitination in response to MMS by distinct mechanisms. This is consistent with a recent report showing that mutation of the INO80 subunits ies3
together with a rsc4
-K25R mutation in RSC results in greater sensitivity to MMS than mutation of either complex alone (57
). Importantly, our data suggest that RSC/PBAF impacts on PCNA loading and ubiquitination more broadly in response to DNA damage.