RNF168-mediated H2A neddylation antagonizes its ubiquitination and regulates DNA damage repair

Abstract

BSTRACTNEDD8 is an important regulatory factor in many biological processes. However, the substrates for neddylation, and the relationship between the ubiquitin and NEDD8 pathways remain largely unknown. Here, we show that NEDD8 is covalently conjugated to histone 2A (H2A), and that neddylation of H2A antagonizes its ubiquitylation. NEDD8 suppresses ubiquitylation of H2A, and a decreased level of free NEDD8 promotes H2A ubiquitylation. Furthermore, we found that the E3 ligase RNF168 promotes both H2A ubiquitylation and neddylation. Interestingly, RNF168 is itself a substrate for NEDD8, and neddylation of RNF168 is necessary for its E3 ubiquitin activity. Inhibition of RNF168 neddylation impairs the interaction between RNF168 and its E2 enzyme Ubc13 (also known as UBE2N). Moreover, in response to DNA damage, the level of H2A neddylation decreased with an increase in the ubiquitylation of H2A, which facilitates DNA damage repair. During the later stages of damage repair, H2A neddylation increased gradually, whereas ubiquitylation decreased to basal levels. Mechanistically, NEDD8 negatively regulates the DNA damage repair process through suppression of the ubiquitylation of H2A and cH2AX, which further blocks the recruitment of the damage response protein BRCA1. Our findings elucidate the relationship of H2A ubiquitylation and neddylation, and suggest a novel modulatory approach to DNA damage repair through the neddylation pathway.

Inhibition of Vpx-Mediated SAMHD1 and Vpr-Mediated Host Helicase Transcription Factor Degradation by Selective Disruption of Viral CRL4 (DCAF1) E3 Ubiquitin Ligase Assembly

“…A neddylationactivating enzyme (NAE) inhibitor, MLN4924, inhibited the neddylation of Vpx-bound Cullin4 and blocked the Vpx-induced degradation of SAMHD1 proteins (60). However, since the neddylation modification is also essential for cellular CRL functions involved in cell cycle control (75,76), signal transduction (77)(78)(79), and cell survival from DNA damage (62,63,80), using the neddylation inhibitor MLN4924 to inhibit viral replication may not be sufficiently selective (64). We now suggest that a highly conserved zinc-binding motif of the viral proteins Vpx and Vpr is responsible for the recruitment of the CRL4 (DCAF1) E3 ligase, and TPEN directly inhibits the binding of Vpx/Vpr and DCAF1 but not the cellular CRL4 E3 ligase assembly and might therefore be less toxic to host cells.…”

“…The neddylation inhibitor MLN4924 blocks Vpx function and preserves the antiviral activity of SAMHD1. However, neddylation is important for normal cellular regulation (62)(63)(64), a consideration that clearly affects its use as an optimal antiviral target.…”

Ubiquitin and ubiquitin-like molecules in DNA double strand break repair

“…RNF111/UBE2M-mediated neddylation inhibits BRCA1 and CtIP-mediated DNA end resection, and regulates the choice between NHEJ and HR and also the balances between different recombination sub-pathways [110]. Interestingly, RNF168 is reported to function as a NEDD8 E3 ligase after DNA damage [111]. It mediates both H2A ubiquitination and neddylation and the two modifications are against each other.…”

Ribosomal protein L6 (RPL6) is recruited to DNA damage sites in a poly(ADP-ribose) polymerase–dependent manner and regulates the DNA damage response

“…The subcellular localization of proteins was examined by immunofluorescence microscopy according to our previous protocol (47) and observed using an LSM 710 NLO and DuoScan (CarlZeiss, Germany).…”