Ubiquitylation, neddylation and the DNA damage response

Abstract

Failure of accurate DNA damage sensing and repair mechanisms manifests as a variety of human diseases, including neurodegenerative disorders, immunodeficiency, infertility and cancer. The accuracy and efficiency of DNA damage detection and repair, collectively termed the DNA damage response (DDR), requires the recruitment and subsequent post-translational modification (PTM) of a complex network of proteins. Ubiquitin and the ubiquitin-like protein (UBL) SUMO have established roles in regulating the cellular response to DNA double-strand breaks (DSBs). A role for other UBLs, such as NEDD8, is also now emerging. This article provides an overview of the DDR, discusses our current understanding of the process and function of PTM by ubiquitin and NEDD8, and reviews the literature surrounding the role of ubiquitylation and neddylation in DNA repair processes, focusing particularly on DNA DSB repair.

Keywords: DNA damage response; MLN4924; NEDD8; double-strand break repair; ubiquitin.

Figure 1.

Simplified illustration of the major protein players involved in ubiquitin signalling following DSB induction. See text for details. Horizontal lines represent DNA. P, phosphorylation; Ub, ubiquitylation; Me, methylation. Protein X denotes unknown protein.

Figure 2.

Illustration of ubiquitylation cascade. Ubiquitin is produced as a precursor polypeptide and cleaved to reveal a carboxyl-terminal GG- motif. In an ATP-dependent reaction, an E1 enzyme transforms this motif into a ubiquitin-adenylate intermediate, which reacts with a Cys in the catalytic domain of the E1 to form an E1∼Ub, thioester linkage. At least for UBA1 (the best-characterized ubiquitin E1), a second ubiquitin molecule is adenylated and remains non-covalently linked to the E1 adenylation active site. Double loading of the E1 with ubiquitin is believed to potentiate transfer of ubiquitin from the E1 to the E2 [99]. The ubiquitin-charged E1 is recognized by an E2 conjugating enzyme and ubiquitin is transferred to the catalytic cysteine of the E2 via a thioester linkage. Ubiquitin is subsequently conjugated to a substrate lysine, through E2 recognition of a substrate/E3 ligase complex. E1 and E3 binding sites to the E2 overlap, ensuring progression of the ubiquitylation cascade. RING E3s facilitate transfer of ubiquitin from the E2 to substrate without binding ubiquitin directly. Alternatively, ubiquitin is transferred to an active site cysteine in HECT/RBR E3s before forming an isopeptide linkage with the substrate lysine. Multiple cycles of substrate binding to ubiquitin-charged E2s lead to ubiquitin chain formation. Ubiquitylation can be reversed by de-ubiquitylating enzymes (DUBs).

Figure 3.

NEDD8 sequence homology and the neddylation cascade. (a) Sequence alignment of human NEDD8, ubiquitin and the NEDD8 homologues in Saccharomyces pombe (ubl1) and Saccharomyces cerevisiae (rub1) using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Lysines critical for ubiquitin chain formation are outlined in blue. Residue 72 (Arg in ubiquitin and Ala in NEDD8) critical for E1 specificity is outlined in red. Colours of amino acids relate to their physiochemical properties: red, small (small + hydrophobic, including aromatic Y); blue, acidic; magenta, basic; green, hydroxyl + sulfhydryl + amine + G. Asterisk (*) denotes fully conserved residue, the symbols :/. denote conservation between groups of strongly/weakly similar properties. (b) Representation of the major neddylation pathway components. NEDD8 (N8) is conjugated in an ATP-dependent cascade involving an E1 (NAE1-UBA3), E2 (UBE2M or F) and E3 (RBX1 or -2) to cullin substrates (sub). The enzymatic pathway is analogous to ubiquitylation (see figure 2 for details). DCUN1D1–5 are cofactors for the NEDD8 E3s. Neddylation is reversed by the CSN complex. MLN4924 inhibits UBA3. See text for details.

Figure 4.

Cullin-RING ligases (CRLs). Simplified diagram of CRL structure. The cullin forms the backbone of the CRL complex. Cullin homology domains at the carboxyl-terminus are required for binding to the E3 and the amino-terminus interacts with the substrate adaptor proteins where required (there is no known substrate adaptor for CUL3) [242]. Neddylation of the cullins on a conserved carboxyl-terminal lysine induces conformational changes to promote ubiquitylation of CRL substrates. SKP1 and DDB1 are the substrate adaptor proteins for CRL1 and CRL4 complexes, respectively. F-box proteins and DCAFs are the substrate receptors for CRL1 and CRL4 complexes, respectively. See text for more details.