PIDD orchestrates translesion DNA synthesis in response to UV irradiation

Abstract

PIDD has been implicated in survival and apoptotic pathways in response to DNA damage, and a role for PIDD was recently identified in non-homologous end-joining (NHEJ) repair induced by c-irradiation. Here, we present an interaction of PIDD with PCNA, first identified in a proteomics screen. PCNA has essential functions in DNA replication and repair following UV irradiation. Translesion synthesis (TLS) is a process that prevents UV irradiation-induced replication blockage and is characterized by PCNA monoubiquitination and interaction with the TLS polymerase eta (polg). Both of these processes are inhibited by p21. We report that PIDD modulates p21-PCNA dissociation, and promotes PCNA monoubiquitination and interaction with polg in response to UV irradiation. Furthermore, PIDD deficiency leads to a defect in TLS that is associated, both in vitro and in vivo, with cellular sensitization to UV-induced apoptosis. Thus, PIDD performs key functions upon UV irradiation, including TLS, NHEJ, NF-jB activation and cell death. Cell Death and Differentiation (2011) 18, 1036-1045 doi:10.1038/cdd.2011; published online 18 March 2011 DNA lesions can result from endogenous metabolic processes as well as from exogenous DNA damaging agents. In both cases, different cellular responses are engaged (cell cycle arrest, repair pathways or apoptosis) to maintain genetic integrity. PCNA (proliferating cell nuclear antigen) acts as a DNA sliding clamp, which helps loading of replicative DNA polymerases. PCNA is involved in several forms of DNA repair, such as NER (nucleotide excision repair), BER (base excision repair) and MMR (mismatch repair), and also in other aspects of DNA metabolism, such as replication, chromatin assembly and cohesion. 1 PCNA mediates these different functions through interactions with proteins specific to each process. 2 Many of these PCNA interacting proteins (PIPs) contain a so-called PIP-box. 1,3 Competition between different partners for the same binding site on PCNA is one of the mechanisms that coordinate the functions of PCNA in DNA replication and repair. PCNA is loaded around the DNA by the conserved chaperone-like clamp loader complex RFC (replication factor C), consisting of five subunits (RFC1 to RFC5). 4,5 The RFC subunits all contain a PIP-box and thereby physically interact with PCNA. 6 Once PCNA is linked to the DNA, the RFC complex is ejected from the clamp to allow DNA polymerase access to the clamp. 7 The protein with the strongest affinity for PCNA is the PIPbox containing p21 (Cip1/Waf1), 8 a potent cyclin-dependent kinase (CDK) inhibitor involved in cell cycle regulation, except in response to UV, where cell cycle arrest is independent of p21. 9 P21 binds to PCNA and inhibits its activity, 10,11 mainly because p21 obstructs the interaction of PCNA with DNA replication and repair factors. [12][13][14][15] In vitro studies have shown that p21 also inhibits the loading of PCNA onto DNA, 7 thereby compromising DNA repair. 9 UV is one of the major exogenous sources of DNA damage. The most common...

Caspase-2: the orphan caspase

“…Tschopp identified a nuclear PIDD complex that contains PCNA (proliferating cell nuclear antigen), an important component of DNA repair machinery. 70 PCNA acts as a DNA sliding clamp that binds DNA polymerase and prevents its dissociation from the template DNA strand. The PIDD-PCNA complex is induced by UV stimulation and appears to facilitate the loading of PCNA onto template DNA.…”

The PIDDosome, DNA-damage-induced apoptosis and beyond

“…4 Large-scale immunoprecipitations with PIDD carried out in two different labs confirmed that most PIDD interactors appeared to be members from the DNA repair machinery and/or the cell cycle checkpoint machinery (C Du and A Villunger, personal communications). 41 Consistent with this, it is known that both PIDD and caspase-2 can be found in the nucleus (constitutively in the case of caspase-2 and upon DNA damage in the case of PIDD 30,37 ) and that caspase-2 is the only caspase identified so far with a nuclear localization signal in its prodomain. 42 Accumulating evidence from over the last few years point out that caspase-2 and PIDD might be implicated in pathways that act much earlier than DNA damage-induced apoptosis and are important for cell cycle control and DNA repair rather than cell death.…”

“…Of note, the relevance of some of these interactions remains to be formerly confirmed. The interaction between PIDD and DNA-PKc was confirmed by Logette et al 41 In addition, they detected almost all members of the proliferating cell nuclear antigen (PCNA)/ RFC/pold complex as PIDD-interacting partners, the PCNA PIDDosome (Table 1), and found a major role for PIDD in the monoubiquitination of PCNA upon DNA damage. PIDD overexpression or UV irradiation and concomitant PIDD induction triggers PCNA monoubiquitination upon release of p21.…”

“…Several protein interactors of PIDD having a role in DNA replication and/or repair were identified (C Du and A Villunger, personal communications). 41 The DNA-PKc PIDDosome, consisting of a complex of both PIDD and caspase-2 with DNA-PKc, revealed a role for PIDD and caspase-2 in nonhomologous end-joining repair mechanisms (C Du and A Villunger, personal communications) ( Table 1). Of note, the relevance of some of these interactions remains to be formerly confirmed.…”

“…Indeed, PIDD-C is the fragment that is involved in pro-survival and repair functions (TLS and NF-kB activation), while PIDD-CC binds RAIDD and initiates caspase-2 activation. 6,12,41 As suggested by the high homology between their cleavages sites, PIDD is cleaved in a manner similar to the nuclear pore proteins Nup98/96, an autoprocessing mechanism also shared by inteins. 6,[58][59][60][61] Characterization of PIDD processing showed that it is sequentially cleaved: in a first cleavage step, PIDD undergoes a cleavage at position S445, generating PIDD-N and PIDD-C, and in a second equivalent cleavage event, PIDD-C is further cleaved at S588 yielding the PIDD-CC fragment 6 ( Figure 2).…”