NEDDylation antagonizes ubiquitination of proliferating cell nuclear antigen and regulates the recruitment of polymerase η in response to oxidative DNA damage

Abstract

NEDDylation has been shown to participate in the DNA damage pathway, but the substrates of neural precursor cell expressed developmentally downregulated 8 (NEDD8) and the roles of NEDDylation involved in the DNA damage response (DDR) are largely unknown. Translesion synthesis (TLS) is a damage-tolerance mechanism, in which RAD18/RAD6-mediated monoubiquitinated proliferating cell nuclear antigen (PCNA) promotes recruitment of polymerase η (polη) to bypass lesions. Here we identify PCNA as a substrate of NEDD8, and show that E3 ligase RAD18-catalyzed PCNA NEDDylation antagonizes its ubiquitination. In addition, NEDP1 acts as the deNEDDylase of PCNA, and NEDP1 deletion enhances PCNA NEDDylation but reduces its ubiquitination. In response to H2O2 stimulation, NEDP1 disassociates from PCNA and RAD18-dependent PCNA NEDDylation increases markedly after its ubiquitination. Impairment of NEDDylation by Ubc12 knockout enhances PCNA ubiquitination and promotes PCNA-polη interaction, while up-regulation of NEDDylation by NEDD8 overexpression or NEDP1 deletion reduces the excessive accumulation of ubiquitinated PCNA, thus inhibits PCNA-polη interaction and blocks polη foci formation. Moreover, Ubc12 knockout decreases cell sensitivity to H2O2-induced oxidative stress, but NEDP1 deletion aggravates this sensitivity. Collectively, our study elucidates the important role of NEDDylation in the DDR as a modulator of PCNA monoubiquitination and polη recruitment.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-017-0455-x) contains supplementary material, which is available to authorized users.

The Balance between Mono- and NEDD8-Chains Controlled by NEDP1 upon DNA Damage Is a Regulatory Module of the HSP70 ATPase Activity

“…The role of ubiquitin and SUMO modification in the DNA-damage response is established at the chromatin level, where upon recruitment they initiate complex signaling events for DNA repair and/or the induction of apoptosis (Jackson and Durocher, 2013;Zhao et al, 2014). Nuclear functions of NEDD8 required for DNA repair have also been reported through NEDDylation of cullins and non-cullin targets, including histones H2A, H4, and PCNA (Li et al, 2014;Ma et al, 2013;Guan et al, 2018). In this study, the combination of C. elegans genetics and biochemistry in human cells reveals a cytoplasmic role for NEDD8, as a protein quality control pathway of the apoptosome formation during the DNA damage-induced apoptosis.…”

Human PCNA Structure, Function and Interactions

“…As different modifications on the same residue are mutually exclusive, dynamic switching between them regulates PCNA's interactions with other proteins. NEDDYlation at K164 antagonizes ubiquitination and regulates the recruitment of Pol η in response to oxidative DNA damage [71]. Methylation at K248 stabilizes PCNA protein levels and enhances PCNA's interaction with FEN1 [72].…”

The NEDD8 cycle controlled by NEDP1 upon DNA damage is a regulatory module of the HSP70 ATPase activity

“…The role of ubiquitin and SUMO modification in the DNA damage response is established at the chromatin level, where upon recruitment they initiate complex signalling events for DNA repair and/or the induction of apoptosis (Jackson and Durocher, 2013;Zhao et al, 2014). Nuclear functions of NEDD8 required for DNA repair have also been reported through NEDDylation of cullins and non-cullin targets, including histones H2A, H4 and PCNA (Li et al, 2014;Ma et al, 2013;Guan et al, 2017). In this study, the combination of C. elegans genetics and biochemistry in human cells reveals a previously uncharacterized cytoplasmic role for NEDD8, as protein quality control pathway of the apoptosome formation during the DNA-damage induced apoptosis.…”