DDB2 (Damaged DNA binding protein 2) in nucleotide excision repair and DNA damage response

Abstract

DDB2 was identified as a protein involved in the Nucleotide Excision Repair (NER), a major DNA repair mechanism that repairs UV damage to prevent accumulation of mutations and tumorigenesis. However, recent studies indicated additional functions of DDB2 in the DNA damage response pathway. Herein, we discuss the proposed mechanisms by which DDB2 activates NER and programmed cell death upon DNA damage through its E3 ligase activity.

Introduction

DDB2 (Damaged-DNA binding protein 2), encoded by the XPE gene, participates in NER.^1–3 It forms a heterodimeric complex with DDB1 and it has been implicated in the recognition of UV-damaged DNA.^4–7 Interestingly while DDB1 is highly evolutionary conserved among eukaryotes, homolog of DDB2 is not found in lower organisms.² This evolutionary conservation of DDB1, but not of DDB2 among species, suggests that DDB1 possesses DDB2-independent functions, and that DDB2 has evolved with the evolutional complexities in higher organisms. The role of DDB2 in the Nucleotide Excision Repair has been discussed in details in previous review articles.^1–3 Therefore, in this review, we focus on the more recent developments on DDB2.

Our laboratory was the first to demonstrate that DDB2, along with DDB1, associates with Cul4A, an E3 ligase component of the ubiquitin-mediated proteasomal degradation pathway.⁸ That observation provided new insights into the function of DDB2 as a substrate-adapter in the Ub-proteasome pathway. The observation that the XPE-mutants of DDB2 failed to bind to Cul4A indicated a role of the Cul4A-DDB2 association in NER. Further support for the role of DDB2 as an adapter protein for an E3 ligase was evidenced when DDB2 was found to be associated with COP9 Signalosome complex (CSN).⁹ CSN shares significant structural homology with the 19S lid of 26S proteasome, and it participates in the ubiquitin-proteasome mediated degradation.^10,11 CSN is known to bind to Cullins and it is believed to play a role in their recycling through its deneddylation activity.^12,13 DDB2 forms a complex with CSN subunits in the absence of DNA damage.⁹ Interestingly, upon UV-irradiation, CSN subunits were shown to be released from DDB2, DDB1, Cul4A, further supporting the notion that E3 ligase activity of DDB2 complex is required for its repair function.⁹

At this point, all proposed models for the NER function of DDB2 are related to the E3 ligase complexes of Cul4A or Cul4B. DDB1 serves as an adapter protein for those interactions. The NER recognition factor XPC was shown to be polyubiquitinated upon DNA damage by Cul4A-DDB1-DDB2 complex in vivo.¹⁴ This posttranslational modification was shown to be dependent on DDB2, as the naturally occurring mutants of DDB2, XP2ROSV and XP82TO are deficient in XPC polyubiquitination upon UV DNA damage.¹⁴ While the precise role of the XPC-modification is not clear, it has been suggested that it is important for the assembly of the repair complex or it serves as a signal for the initiation of NER.

Two groups suggested that the DDB2-associated Cul4-complexes mono-ubiquitinate histones on the damaged chromatin to re-model its structure, required for the recruitment of the NER proteins.^15,16 For instance, one study indicated that DDB2 functions as an adapter protein for mono-ubiquitination of histone H2A by the Cul4A-DDB1 ligase and suggested a role for H2A ubiquitination in the NER.¹⁵ Other study showed that histones H3 and H4 were ubiquitinated by DDB2-containing ligase, and the ubiquitination was induced by UV irradiation. Moreover, complexes composed of Cul4A/B, DDB1, DDB2 and Roc1 were required for ubiquitination of H3 and H4.¹⁶ Ubiquitination of H3/H4 was proposed to be important for recruiting the NER recognition factor XPC to UV-damaged chromatin.¹⁶ Knockdown of Cul4A prevented recruitment of XPC to the DNA damage sites resulting in deficiency of CPD removal.¹⁶ Moreover, the Cul4A knockout MEFs exhibited impaired DNA repair, as measured by UDS.¹⁷ While the studies mentioned above provided evidence of the role for Cul4-DDB1-DDB2 in the ubiquitination of histones and XPC, direct evidence for a role of these ubiquitinations in NER is not available.

DDB2 is itself a target of Cul4A.¹⁸ Degradation of DDB2 was observed also in UV-irradiated cells within hours following UV-irradtiation.^19,20 Also, DDB2 is tightly bound to the chromatin shortly after UV.²¹ Those two events (chromatin association and degradation) were observed specifically in UV-irradiated cells. IR did not affect the level of DDB2.²¹ It is noteworthy that IR-induced c-Abl kinase was shown to disrupt the interaction between DDB2 and DDB1.²² Nevertheless, DDB2 proteolysis at the DNA damage sites was implicated also in NER because silencing Cul4A gives rise to a delay in CPDs removal from the DNA damage site.¹⁹ Because Cul4A siRNA also prevented the recruitment of XPC to the DNA damage sites, it was suggested that removal of DDB2 from the damaged DNA by Cul4A mediated degradation might be important for the recruitment of XPC.¹⁹ Chen et al. on the other hand, proposed an opposite view.²³ These authors studied the role of c-Abl in regulating the function of DDB2. They showed that c-Abl enhanced both polyubiquitination and proteolysis of DDB2 by activating Cul4A. Moreover, those authors correlated the enhanced proteolysis of DDB2 to inhibition of NER by the c-Abl proto-oncoprotein. In another study, Zotter et al. investigated the rate of XPG recruitment at the damaged chromatin.²⁴ XPG is recruited by XPA, which in turn depends upon the recognition of damaged chromatin by XPC. Surprisingly, those authors did not see any difference in the rate of XPG recruitment between DDB2-proficient and DDB2-deficient cells. Their results argue against the role of DDB2 in the early, recognition step of NER.

Using a mouse knockout model for DDB2 we observed genetic evidence that DDB2 participates in NER by regulating the levels of p21^Waf1/Cip1.²⁵ In addition, we discovered a significant role of the DDB2-mediated regulation of p21^Waf1/Cip1 in the DNA damage response.^25,26 Those observations are summarized below.

Degradation of p53^S18P by Cul4-DDB1-DDB2 Ligase

We found that DDB2 participates in NER indirectly by regulating the DNA damage response pathway. Upon DNA damage, Ataxia telangiectasia mutated kinase (ATM) and ataxia telangiectasia RAD3-related kinase (ATR) are activated and are recruited to the damaged-chromatin. The activated ATM/ATR phosphorylate p53 at Ser15 in humans, Ser18 in mouse, Chk1 and Chk2, which further phosphorylate p53 at Ser20.^27,28 Phosphorylation at residue Ser18 in mouse p53 does not contribute to the stability of p53, but it rather stimulates its transcriptional activity.²⁹ p53 plays a central role in cell cycle arrest upon DNA damage. It ensures that the cells will not proceed into the cell cycle before the damage is repaired. G₁ phase arrest of the cell cycle is very important for the genomic integrity since it prevents entry into S phase and it does not allow replication of damaged DNA, avoiding accumulation of mutations.

We found that in low-dose UV-irradiated mouse embryonic fibroblasts (MEFs), DDB2 plays an important role in regulating the cellular levels of p53^S18P, but not the levels of total p53. We showed that DDB2 induced degradation of p53^S18P through the ubiquitin-proteasome pathway.²⁵ We provided evidence that the proteolysis involved Cul4A and DDB1. Moreover, we observed an interaction between DDB2 and p53^S18P but to a much lower extent in comparison to the interaction between DDB1 and p53^S18P.²⁵ While we suggested that DDB2 participates in the proteolysis of p53^S18P by increasing nuclear accumulation of DDB1, we did not rule out the possibility that DDB2 also participates in targeting p53^S18P for proteasomal degradation.

Interestingly, we did not detect any difference in p53^S18P between the wild type and DDB2 (−/−) MEFs upon high-dose of UV (50J/m²) and cisplatin treatment.²⁶ DDB2 plays differential roles upon high and low-dose of UV irradiation. At high-dose of UV, when the DNA damage is too high to be repaired, we suggest that p53 is stabilized due to further modifications on its N terminus and DDB2 does not participate in its degradation. One of the transcriptional target of p53 is p21^Waf1/Cip1, a cyclin-dependent kinase inhibitor.³⁰ We provided evidence that indeed accumulation of p53^S18P in the DDB2-deficient cells leads to higher transcriptional activity and causes increased expression of p21^Waf1/Cip1 in human and in mouse cells upon low-dose of UV.²⁵

DDB2 Activates NER by Regulating p21^Waf1/Cip1

p21^Waf1/Cip1 interacts with proliferation cell nuclear antigen (PCNA) and inhibits NER both in vitro and in vivo.³¹ PCNA acts as a DNA clamp required for recruitment of DNA polymerase δ onto the DNA, thus it is indispensable for DNA synthesis. p21^Waf1/Cip1 can block DNA synthesis by DNA polymerase δ through a direct interaction with PCNA.^32–34 Two studies indicated that an elevated level of p21^Waf1/Cip1 inhibits NER through inhibition of PCNA.^31,35 We demonstrated that high level of p21^Waf1/Cip1 is the cause of NER deficiency in DDB2(−/−) cells.²⁵ Deletion or knockdown of p21^Waf1/Cip1 reverses NER-deficient phenotype in DDB2(−/−) background, measured by unscheduled DNA synthesis (Fig. 1A). Our observations provided genetic evidence linking the regulation of p21^Waf1/Cip1 to the NER activity of DDB2 (Fig. 1A).

Figure 1.

DDB2 decides cell fate upon DNA damage. (A) DDB2 activates Nucleotide Excision Repair by regulating p21^Waf1/Cip1. In low-dose UV-irradiated cells DDB2 regulates p21^Waf1/Cip1 both at the level of transcription and proteolysis. By keeping the levels of p21^Waf1/Cip1, DDB2 ensures efficient DNA Repair DNA synthesis and completes NER. (B) Upon high-dose of UV-irradiation or treatment with other DNA damaging agents, when the DNA damage is irreparable, DDB2 downregulates p21^Waf1/Cip1 by proteolysis, allowing the cells to undergo apoptosis.

DDB2 Induces Proteolysis of p21^Waf1/Cip1 After DNA Damage

DDB2 regulates p21^Waf1/Cip1 both, at the level of RNA through proteolysis of p53^S18P and at protein level by degradation through the Ub-proteasome pathway (Fig. 1A). DDB2-regulation of p21^Waf1/Cip1 at transcriptional level takes place only upon low-dose of UV.^25,26 However, the involvement of DDB2 in the proteolysis of p21^Waf1/Cip1 was observed under all conditions. DDB2 binds to p21^Waf1/Cip1, inducing its polyubiquitination. When DDB2 was silenced or deleted, ubiquitination of p21^Waf1/Cip1 was inhibited, causing it to accumulate at a high level. We speculate that DDB2 serves as an adapter protein linking p21^Waf1/Cip1 to an E3 ligase complex, such as Cul4-DDB1.

p21^Waf1/Cip1 protein is degraded by both ubiquitin dependent and independent mechanisms.^36–40 We exclude the possibility that DDB2 participates in the ubiquitin independent degradation of p21^Waf1/Cip1, since polyubiquitination of p21^Waf1/Cip1 was robustly inhibited upon DDB2 silencing.

Recently, CRL4^Cdt2 E3 ligase (composed of the Cul4A/B, DDB1 and the DCAF subunit Cdt2) was found to proteolyse p21^Waf1/Cip1 upon exposure to low-dose of UV.^39,41 That pathway involves PCNA. Depletion of Cul4A/4B or PCNA by siRNA not only prevented the UV induced proteasomal degradation of p21^Waf1/Cip1, but also resulted in the accumulation of p21^Waf1/Cip1 in the S phase of the cell cycle. Further studies will be necessary to determine the overlap between the DDB2- and Cdt2-pathway of p21^Waf1/Cip1 proteolysis. It will be important to determine the subcellular compartment for p21^Waf1/Cip1 degradation by DDB2. We speculate that it might be on the chromatin. Recent studies indicated that the replication licensing factor Cdt1 is recruited to the chromatin bound PCNA via PIP box (PCNA-binding motif).⁴² Upon recruitment to the chromatin, Cdt1 is polyubiquitinated by Cul4A/DDB1/Cdt2 complex and it is targeted for proteasomal degradation.⁴² p21^Waf1/Cip1 contains similar PIP motif within its sequence and PIP p21^Waf1/Cip1 peptide is sufficient to recruit Cul4A/Cdt2/DDB1 to the chromatin.⁴² The same study also suggested a role of chromatin in the E3 ligase function of Cul4. It is possible that the histone-ligase function of DDB2 plays a role in the ubiquitination of p21^Waf1/Cip1.

DDB2-p21^Waf1/Cip1 Axis Regulates Apoptosis After DNA Damage

It has been previously shown that DDB2 (−/−) MEFs are deficient in apoptosis.⁴³ Cells lacking DDB2 are impaired in apoptosis induced not only by high-dose of UV-irradiation, but also by cisplatin, aclarubicin (two drugs commonly used in chemotherapy), ionizing radiation and E2F1 overexpression.²⁶ Under condition of extreme DNA damage, p53 induces apoptosis, which functions as a mechanism against tumor development. p53 triggers the intrinsic apoptotic pathway through multiple mechanisms.⁴⁴ For example, p53 induces expression of proapoptotic genes Bax, PUMA and NOXA, members of the Bcl-2 family.^45,46 Besides the transcriptional-dependent function, p53 may play a direct role in activating the intrinsic apoptotic pathway through translocation to the mitochondria. p53 may promote apoptosis by induction of cytochrome c release.⁴⁴ Interestingly, all those p53-functions are active in the DDB2(−/−) MEFs, but the cells fail to undergo apoptosis.²⁶

The accumulation of p21^Waf1/Cip1 in the absence of DDB2 is much more pronounced after high-dose of UV, cisplatin and aclarubicin in comparison to low-dose of UV.^25,26 Several studies suggested an important role of p21^Waf1/Cip1 in inhibition of apoptosis.^47,48 Also, it was shown that depletion of p21^Waf1/Cip1 sensitized cells to DNA damage induced apoptosis.^49,50 Thus, p21^Waf1/Cip1 functions as a barrier and an inhibitor of apoptosis after DNA damage. One of the proposed indirect mechanisms by which p21^Waf1/Cip1 protects cells from apoptosis is through its cell cycle inhibitory function. Blocking cyclinA/cdk2 complex by p21^Waf1/Cip1 leads to consequent resistance to programmed cell death.

Instead of undergoing apoptosis, DDB2 deficient cells exhibited cell cycle delay, which seems to be a dominant event coinciding with the very high levels of p21^Waf1/Cip1. In DDB2(−/−) MEFs, cisplatin and aclarubicin caused an increase in the G₁ population, whereas UV irradiation resulted in an increase in the population of S phase cells and, consequently, to S phase delay. In addition, HeLa cells expressing DDB2 shRNA exhibited S phase delay. Thus, DDB2 is required for apoptosis following DNA damage and in the absence of DDB2, cells undergo cell cycle arrest. Resistance to apoptosis of the DDB2-deficient cells is due to accumulation of p21^Waf1/Cip1 because deletion of p21^Waf1/Cip1 restored the apoptotic response.

Efficient inhibition of Cdk2 by p21^Waf1/Cip1 requires Mdm2.⁵¹ The authors of that study showed that cells exhibited inefficient inhibition of Cdk2 when depleted with Mdm2-siRNA compared to that when treated with nutlin-3, a selective inhibitor of p53-Mdm2 interaction. The mechanism by which Mdm2 participates in Cdk-inhibition by p21^Waf1/Cip1 remains unclear. The observation, however, is consistent with a previous study that demonstrated existence of cell cycle inhibitory domains in Mdm2.^52,53 We found that Mdm2 is required for p21^Waf1/Cip1-induced cell cycle delay that inhibits apoptosis. Depletion of Mdm2 (by siRNA) in DDB2-deficient cells restored apoptosis. We provided further evidence that cdk2 activity is required for sensitizing cells to apoptosis.²⁶

We found DDB2 as a key determinant in deciding cell fate following DNA damage. We propose that upon low-dose of UV irradiation, DDB2 regulates NER indirectly through termination of DNA damage response pathway by targeting p53^S18P and p21^Waf1/Cip1 for proteasomal degradation, allowing the NER to take place efficiently and completely (Fig. 1A). When the DNA damage cannot be repaired, DDB2 downregulates p21^Waf1/Cip1 through ubiquitin-mediated proteasomal degradation, which is critical for the cells to undergo apoptosis (Fig. 1B). This model provides a new insight for DDB2 function. In addition to DNA repair, DDB2 plays an important role as a molecular switch in deciding cell fate (apoptosis or arrest) upon DNA damage (Fig. 1B).