Post-incision events induced by UV : regulation of incision and the role of post-incision factors in mammalian NER
Overmeer, R.M.
Citation
Overmeer, R. M. (2010, September 29). Post-incision events induced by UV : regulation of incision and the role of post-incision factors in mammalian NER.
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Perspectives
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Chapter 3: Perspectives
From old observations to novel insights
Recent experiments have shed new light on old observations. Novel insights in coordination of NER mediated incision with gapfi lling provide a mechanistic explanation for the inhibi- tion of NER when ligation is impaired, observations made more than two decades ago. The central role of RPA in this regulation raises interesting questions; most notably, how can it be that such an abundant protein can be rate limiting? An obvious answer would lie in post translational modifi cations of RPA infl uencing its affi nity for different structures. Phospho- rylation of RPA has been shown to be involved in response to cisplatinum, bleomycin and UV (Anantha et al., 2007; Rodrigo et al., 2000). This phosphorylation was shown however to be most prevalent during replication (Vassin et al., 2009) and mainly activated by DNA damage mediated inhibition of replication (Patrick et al., 2005; Binz et al., 2003; Rodrigo et al., 2000). Parallel to the ATR dependent phosphorylation of RPA, RPA is released from the PML bodies in an ATR dependent manner most likely to generate additional free RPA for NER (Park et al., 2005; Barr et al., 2003). Interestingly, ATR only affects NER during the S phase (Auclair et al., 2008). Taken into account that RPA is also abundant during G1 (Din et al., 1990), one would expect a level of regulation reminiscent of that found during S phase.
Since ATR defi ciency does not affect incision control by RPA outside of S phase, regulation by phosphorylation seems unlikely; instead, one could envision that this regulation is media- ted through an as of yet undefi ned modifi cation. We speculate that SUMOylation might be a good candidate based on two observations: the interaction of RPA with PML bodies (which are known to contain SUMOylated proteins) and the emergence of RPAp70 in a screen for SUMOylated proteins in cells treated by proteosome inhibitors (Schimmel et al., 2008). As an alternative mechanism we speculate that RPA interacting proteins could play a role in the regulation of RPA activity. An RPA interacting protein such as RIPβ (RPA interacting protein β) could mediate RPA’s substrate specifi city and/ or activity (Park et al., 2005).
The necessity of ligation to initiate novel incisions on one hand and the involvement of chromatin remodelers for chromatin restoration on the other lead to the question whether there is an additional feedback loop, in which restoration of the chromatin structure can infl uence upstream events. Such regulation is not unconceivable as post-incision factors such as PCNA have been shown to interact with chromatin remodelers such as chromatin assembly factor 1 (CAF1) (Green and Almouzni, 2003). In addition post-incision factors remain associated with the chromatin at sites of NER even when gapfi lling and ligation have been completed (Overmeer et al., 2010a; Overmeer et al., 2010b). Momentarily the role of chromatin remo- delers in the activity and heterogeneity of repair pathways such as NER is a hot topic. It will therefore be interesting to examine whether a coupling exists between chromatin restoration and initiation events and whether the prolonged association of post-incision factors is functi- onal or merely due to ineffi cient unloading of PCNA leading to non-functional association of interacting factors. To answer such questions one could inhibit chromatin remodelling, most
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conveniently by knockdown of (multiple) factors using a siRNA strategy (Polo et al., 2006).
A regulatory loop would be manifested by an inhibition of photolesion removal or repair synthesis, whilst the requirement of the post-incision factors in chromatin remodelling would lead to an increased accumulation of such factors in time and intensity, similar to that seen for pre-incision factors when repair replication is inhibited (Overmeer et al., 2010b). Such a regulatory loop seems unlikely as knockdown of CAF1 had no effect on lesion removal and minor effects on PCNA recruitment and repair synthesis (Polo et al., 2006). However, there may be some redundancy in chromatin remodelers and the potential increase in post-incision factor accumulation was not studied. Moreover the cells studied were non-replicating cells within an asynchronous population and would therefore be expected to have higher levels of replication proteins than quiescent cells (Moser et al., 2007; van der Kuip et al., 1999). In a broader context, inappropriate or incomplete restoration of chromatin structure at the site of NER might provide a signal to prevent quiescent cells entering the cell cycle after UV expo- sure (Cohn et al., 1984) possibly induced by the increased expression of cyclin E (Stubbert et al., 2009).
Reinterpreting dynamic data
The dynamic properties of many proteins have been determined using GFP-tagged proteins and it has been assumed that the residence time could be used as a measure for the time required to participate in the corresponding complex. However, a recent comparison of the association kinetics of endogenous RFCp140 and the dynamics of association of GFP-tagged RFCp140 with DNA damage under conditions that abrogate incision, has forced us to re- consider such a view. It was shown that under conditions of inhibition of repair synthesis, RFCp140 remained present at repair sites for several hours, whilst the residence time of a GFP-tagged RFCp140 was ~70 seconds, only slightly longer than in complexes formed under repair profi cient conditions (~50 seconds). This apparent contradiction between endo- genous and GFP tagged proteins leads to a model in which the complex as a whole remains associated to the site of repair (substrate) whereas the individual proteins continuously asso- ciate and dissociate (Overmeer et al., 2010a). This situation will last as long as the substrate exists. Hence, the residence time of proteins in active complexes is determined by the relative affi nity (dictating the association and dissociation) and the lifespan of a complex (as most complexes dissolve after accomplishing their goal).
The way one interprets the dynamic properties of proteins becomes all the more impor- tant when one tries to fi t such data to mathematical models as was done recently (Dinant et al., 2009; Politi et al., 2005). A relative short association time could either lead to the con- clusion that the factor is one of the last to incorporate into a specifi c complex or it could lead to the conclusion that the factor has a low affi nity for that complex. Without dismissing the studies performed, a much more complete image of complex assembly and incision is expec- ted when dynamic properties of NER factors in a repair profi cient context are compared with those observed under conditions of inhibited incision or gapfi lling. Dynamic parameters for
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pre-incision proteins in NER profi cient cells depend on two factors i.e. the time required for incision and subsequent dissolution of the complex; and the relative affi nity of the proteins for the pre-incision complex (defi ned by the relation between association and dissociation of proteins). Two types of incision defi cient pre-incision complexes can be formed (i.e. with and without RPA). In NER defi cient XP-A, XP-F and XP-G cells, pre-incision factors as- semble in stable (incision defi cient) pre-incision complexes (Overmeer et al., 2010b) unable to associate with other photolesions. In the presence of DNA synthesis inhibitors, unsta- ble pre-incision complexes lacking RPA are formed (Overmeer et al., 2010b). Therefore the measurement of the dynamics of pre-incision factors under conditions where the complex is stably stalled (such as found in NER defi cient cells i.e. unable to incise yet containing RPA) would give an accurate estimate of the relative affi nity of proteins for these stable complexes making it possible to more accurately calculate the time required for incision. In addition by comparing the dynamics of pre-incision proteins in such stable complexes with their dyna- mics in the unstable (RPA lacking) complexes, information can be obtained on how RPA sta- bilizes the pre-incision complex. A similar strategy can be taken to estimate the time required for gapfi lling determining the residence time under conditions of inhibited gapfi lling/ ligation or complete gapfi lling.
Unexpected mechanisms
In addition to shining new light on old observations, recent studies have also revealed (im- plications for) new and unexpected mechanisms. RFC is required for the loading of PCNA and has been shown (at least in vitro) to dissociate after loading PCNA (Podust et al., 1998).
However, the recently observed prolonged association of RFC with NER complexes in the presence of DNA synthesis inhibitors revealed that RFC probably has an additional role in NER (Overmeer et al., 2010a). Interestingly, the in vitro observations used an N-terminally truncated RFCp140, a deletion that has been shown to be dispensable for PCNA loading (Uhlmann et al., 1997). However, the N-terminal region contains a BRCT domain which preferentially binds the 5’phosphate of double stranded DNA (Kobayashi et al., 2006). Taken together these data suggest that RFC has an additional role after loading PCNA and that this role could be to associate with the 5’phosphate to inhibit Rad17 mediated signalling. Alter- natively, the association of RFC at the 5’phosphate places it in the ideal position to unload PCNA, a process that had largely been ignored. Another interesting observation is that the recruitment of the polymerases requires more than (ubiquitinated) PCNA, as evidenced by the requirement of RFC1 for polδ recruitment and XRCC1 for polκ recruitment (Ogi et al., 2010; Overmeer et al., 2010a). In addition recruitment of polε to sites of repair requires CHTF18 whilst DNA repair synthesis seemed unaffected by knockdown of CHTF18 (Ogi et al., 2009). The requirement of these factors in recruiting specifi c polymerases indicates that their recruitment in repair is more regulated than thought. As such, the role of RFC and RFC- like proteins in the recruitment and switching of polymerases and/ or clamps is all the more interesting. Future research should show whether the requirement of factors such as RFC1,
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XRCC1 and CHTF18 for polymerase recruitment is limited to repair synthesis, extends to (translesion) synthesis during replication or holds for exceptional conditions associated with Go state of cells. On the other hand the continued association of clamploaders at sites of repair after loading PCNA suggests additional roles for factors thought to be solely involved in loading PCNA. In particular, the role of Rad17 in signalling with the possible inhibition of thereof by RFC opens up a role for the clamploaders in initiating or inhibiting signalling.
Next to unexpected roles of clamploaders in the recruitment of the polymerases to sites of repair the actual requirement of multiple low fi delity (mutagenic) polymerases is very remar- kable for a repair process that is thought to be error free. It raises questions such as why and what are the cells recruiting mutagenic polymerases for and whether NER is indeed error free under all circumstances. Whereas the relatively high activity of polκ under conditions with low nucleotide concentrations could explain its requirement for effi cient NER (Godoy et al., 2006; Ogi and Lehmann, 2006), the requirement of other mutagenic polymerases is less readily explained. Study the basis of their requirement is made all the more interesting by the apparent division of gapfi lling into 2 parallel, non-redundant sub-pathways (Ogi et al., 2010).
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