Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers.

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs) orchestrates and regulates cellular responses to DSBs at multiple levels, often involving extensive crosstalk between these modifications. Recent findings have revealed compelling insights into the complex mechanisms by which ubiquitin and UBLs regulate protein interactions with DSB sites to promote accurate lesion repair and protection of genome integrity in mammalian cells. These advances offer new therapeutic opportunities for diseases linked to genetic instability.

SUMOylation promotes protective responses to DNA-protein crosslinks.

DNA-protein crosslinks (DPCs) are highly cytotoxic lesions that obstruct essential DNA transactions and whose resolution is critical for cell and organismal fitness. However, the mechanisms by which cells respond to and overcome DPCs remain incompletely understood. Recent studies unveiled a dedicated DPC repair pathway in higher eukaryotes involving the SprT-type metalloprotease SPRTN/DVC1, which proteolytically processes DPCs during DNA replication in a ubiquitin-regulated manner. Here, we show that chemically induced and defined enzymatic DPCs trigger potent chromatin SUMOylation responses targeting the crosslinked proteins and associated factors. Consequently, inhibiting SUMOylation compromises DPC clearance and cellular fitness. We demonstrate that ACRC/GCNA family SprT proteases interact with SUMO and establish important physiological roles of Caenorhabditis elegans GCNA-1 and SUMOylation in promoting germ cell and embryonic survival upon DPC formation. Our findings provide first global insights into signaling responses to DPCs and reveal an evolutionarily conserved function of SUMOylation in facilitating responses to these lesions in metazoans that may complement replication-coupled DPC resolution processes.

FACT subunit Spt16 controls UVSSA recruitment to lesion-stalled RNA Pol II and stimulates TC-NER.

Transcription-coupled nucleotide excision repair (TC-NER) is a dedicated DNA repair pathway that removes transcription-blocking DNA lesions (TBLs). TC-NER is initiated by the recognition of lesion-stalled RNA Polymerase II by the joint action of the TC-NER factors Cockayne Syndrome protein A (CSA), Cockayne Syndrome protein B (CSB) and UV-Stimulated Scaffold Protein A (UVSSA). However, the exact recruitment mechanism of these factors toward TBLs remains elusive. Here, we study the recruitment mechanism of UVSSA using live-cell imaging and show that UVSSA accumulates at TBLs independent of CSA and CSB. Furthermore, using UVSSA deletion mutants, we could separate the CSA interaction function of UVSSA from its DNA damage recruitment activity, which is mediated by the UVSSA VHS and DUF2043 domains, respectively. Quantitative interaction proteomics showed that the Spt16 subunit of the histone chaperone FACT interacts with UVSSA, which is mediated by the DUF2043 domain. Spt16 is recruited to TBLs, independently of UVSSA, to stimulate UVSSA recruitment and TC-NER-mediated repair. Spt16 specifically affects UVSSA, as Spt16 depletion did not affect CSB recruitment, highlighting that different chromatin-modulating factors regulate different reaction steps of the highly orchestrated TC-NER pathway.

UVSSA and USP7, a new couple in transcription-coupled DNA repair.

Transcription-coupled nucleotide excision repair (TC-NER) specifically removes transcription-blocking lesions from our genome. Defects in this pathway are associated with two human disorders: Cockayne syndrome (CS) and UV-sensitive syndrome (UVSS). Despite a similar cellular defect in the UV DNA damage response, patients with these syndromes exhibit strikingly distinct symptoms; CS patients display severe developmental, neurological, and premature aging features, whereas the phenotype of UVSS patients is mostly restricted to UV hypersensitivity. The exact molecular mechanism behind these clinical differences is still unknown; however, they might be explained by additional functions of CS proteins beyond TC-NER. A short overview of the current hypotheses addressing possible molecular mechanisms and the proteins involved are presented in this review. In addition, we will focus on two new players involved in TC-NER which were recently identified: UV-stimulated scaffold protein A (UVSSA) and ubiquitin-specific protease 7 (USP7). UVSSA has been found to be the causative gene for UVSS and, together with USP7, is implicated in regulating TC-NER activity. We will discuss the function of UVSSA and USP7 and how the discovery of these proteins contributes to a better understanding of the molecular mechanisms underlying the clinical differences between UVSS and the more severe CS.

An immunoaffinity purification method for the proteomic analysis of ubiquitinated protein complexes.

Protein ubiquitination plays an important role in the regulation of many cellular processes, including protein degradation, cell cycle regulation, apoptosis, and DNA repair. To study the ubiquitin proteome we have established an immunoaffinity purification method for the proteomic analysis of endogenously ubiquitinated protein complexes. A strong, specific enrichment of ubiquitinated factors was achieved using the FK2 antibody bound to protein G-beaded agarose, which recognizes monoubiquitinated and polyubiquitinated conjugates. Mass spectrometric analysis of two FK2 immunoprecipitations (IPs) resulted in the identification of 296 FK2-specific proteins in both experiments. The isolation of ubiquitinated and ubiquitination-related proteins was confirmed by pathway analyses (using Ingenuity Pathway Analysis and Gene Ontology-annotation enrichment). Additionally, comparing the proteins that specifically came down in the FK2 IP with databases of ubiquitinated proteins showed that a high percentage of proteins in our enriched fraction was indeed ubiquitinated. Finally, assessment of protein-protein interactions revealed that significantly more FK2-specific proteins were residing in protein complexes than in random protein sets. This method, which is capable of isolating both endogenously ubiquitinated proteins and their interacting proteins, can be widely used for unraveling ubiquitin-mediated protein regulation in various cell systems and tissues when comparing different cellular states.

UV-sensitive syndrome protein UVSSA recruits USP7 to regulate transcription-coupled repair.

Transcription-coupled nucleotide-excision repair (TC-NER) is a subpathway of NER that efficiently removes the highly toxic RNA polymerase II blocking lesions in DNA. Defective TC-NER gives rise to the human disorders Cockayne syndrome and UV-sensitive syndrome (UV(S)S). NER initiating factors are known to be regulated by ubiquitination. Using a SILAC-based proteomic approach, we identified UVSSA (formerly known as KIAA1530) as part of a UV-induced ubiquitinated protein complex. Knockdown of UVSSA resulted in TC-NER deficiency. UVSSA was found to be the causative gene for UV(S)S, an unresolved NER deficiency disorder. The UVSSA protein interacts with elongating RNA polymerase II, localizes specifically to UV-induced lesions, resides in chromatin-associated TC-NER complexes and is implicated in stabilizing the TC-NER master organizing protein ERCC6 (also known as CSB) by delivering the deubiquitinating enzyme USP7 to TC-NER complexes. Together, these findings indicate that UVSSA-USP7­mediated stabilization of ERCC6 represents a critical regulatory mechanism of TC-NER in restoring gene expression.

Nucleotide excision repair-induced H2A ubiquitination is dependent on MDC1 and RNF8 and reveals a universal DNA damage response.

Chromatin modifications are an important component of the of DNA damage response (DDR) network that safeguard genomic integrity. Recently, we demonstrated nucleotide excision repair (NER)-dependent histone H2A ubiquitination at sites of ultraviolet (UV)-induced DNA damage. In this study, we show a sustained H2A ubiquitination at damaged DNA, which requires dynamic ubiquitination by Ubc13 and RNF8. Depletion of these enzymes causes UV hypersensitivity without affecting NER, which is indicative of a function for Ubc13 and RNF8 in the downstream UV-DDR. RNF8 is targeted to damaged DNA through an interaction with the double-strand break (DSB)-DDR scaffold protein MDC1, establishing a novel function for MDC1. RNF8 is recruited to sites of UV damage in a cell cycle-independent fashion that requires NER-generated, single-stranded repair intermediates and ataxia telangiectasia-mutated and Rad3-related protein. Our results reveal a conserved pathway of DNA damage-induced H2A ubiquitination for both DSBs and UV lesions, including the recruitment of 53BP1 and Brca1. Although both lesions are processed by independent repair pathways and trigger signaling responses by distinct kinases, they eventually generate the same epigenetic mark, possibly functioning in DNA damage signal amplification.