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The SLX4 Fanconi anemia protein is a tumor suppressor that may act as a key regulator that engages the cell into specific genome maintenance pathways. Here, we show that the SLX4 complex is a SUMO E3 ligase that SUMOylates SLX4 itself and the XPF subunit of the DNA repair/recombination XPF-ERCC1 endonuclease. This SLX4-dependent activity is mediated by a remarkably specific interaction between SLX4 and the SUMO-charged E2 conjugating enzyme UBC9 and relies not only on newly identified SUMO-interacting motifs (SIMs) in SLX4 but also on its BTB domain. In contrast to its ubiquitin-binding UBZ4 motifs, SLX4 SIMs are dispensable for its DNA interstrand crosslink repair functions. Instead, while detrimental in response to global replication stress, the SUMO E3 ligase activity of the SLX4 complex is critical to prevent mitotic catastrophe following common fragile site expression.

Structure-specific DNA endonucleases have critical roles during DNA replication, repair and recombination, yet they also harbor the potential for causing genome instability. Controlling these enzymes may be essential to ensure efficient processing of ad hoc substrates and to prevent random, unscheduled processing of other DNA structures, but it is unknown whether structure-specific endonucleases are regulated in response to DNA damage. Here, we uncover DNA damage-induced activation of Mus81-Eme1 Holliday junction resolvase in fission yeast. This novel regulation requires both Cdc2CDK1 and Rad3ATR-dependent phosphorylations of Eme1. Mus81-Eme1 activation prevents gross chromosomal rearrangements in cells lacking the BLM-related DNA helicase Rqh1. We propose that linking Mus81-Eme1 DNA damaged-induced activation to cell cycle progression ensures efficient resolution of Holliday junctions that escape dissolution by Rqh1-TopIII while preventing unnecessary DNA cleavages.

UV-damaged-DNA-binding protein (UV-DDB) is a heterodimer comprised of DDB1 and DDB2 and integrated in a complex that includes a ubiquitin ligase component, cullin 4A, and Roc1. Here we show that the ubiquitin ligase activity of the DDB2 complex is required for efficient global genome nucleotide excision repair (GG-NER) in chromatin. Mutant DDB2 proteins derived from xeroderma pigmentosum group E patients are not able to mediate ubiquitylation around damaged sites in chromatin. We also found that CSN, a negative regulator of cullin-based ubiquitin ligases, dissociates from the DDB2 complex when the complex binds to damaged DNA and that XPC and Ku oppositely regulate the ubiquitin ligase activity, especially around damaged sites. Furthermore, the DDB2 complex-mediated ubiquitylation plays a role in recruiting XPA to damaged sites. These findings shed some light on the early stages of GG-NER.Nucleotide excision repair (NER) is a principal pathway that removes a variety of helix-distorting DNA lesions, such as the cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidinepyrimidone photoproducts (6-4PPs) generated by UV light (14). In addition, NER can repair with different efficiencies the lesions formed by cisplatin, polycyclic carcinogens, psoralens, and other chemical compounds (46,60). There are two subpathways of NER: global genome NER (GG-NER) and transcription-coupled NER (TC-NER). GG-NER eliminates DNA lesions over the entire genome, whereas TC-NER rapidly removes lesions on the transcribed strands of transcriptionally active genes (18, 51).Various hereditary diseases involve defects in NER, including xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy (TTD), and UV-sensitive syndrome (UV s S) (20,30). XP is characterized by hypersensitivity to sunlight and an increased incidence of sunlight-induced skin cancers (30). CS is also characterized by photosensitivity of the skin, but CS patients have no predisposition to UV-induced skin cancers. Instead, they exhibit severe developmental and neurological abnormalities, as well as premature aging (37). Genetic complementation analysis has defined seven complementation groups in XP (XP-A to XP-G) and two in CS (CS-A and CS-B) (61). It is worth noting that some XP patients exhibit features of CS in addition to symptoms of XP (XP-B/CS, XP-D/CS, and XP-G/CS) and that all gene products responsible for these disorders are integrated in the XPG-transcription factor IIH (TFIIH) complex, suggesting a close relationship between transcription and NER (24).The two pathways differ primarily in how damaged DNA is first recognized (14). In GG-NER, the XPC-HR23B-centrin2 complex and UV-damaged-DNA-binding protein (UV-DDB), and in TC-NER, the RNA polymerase II stalled at a lesion on the transcribed strand, play a role in the initial recognition process (45, 49, 55). These are followed by NER reactions common to both pathways. TFIIH, XPG, XPA, and replication protein A (RPA) are recruited to the lesion, leading to the local unwinding of the DNA double helix. The...

The xeroderma pigmentosum group A protein (XPA) is a core component of nucleotide excision repair (NER). To coordinate early stage NER, XPA interacts with various proteins, including replication protein A (RPA), ERCC1, DDB2, and TFIIH, in addition to UV-damaged or chemical carcinogendamaged DNA. In this study, we investigated the effects of mutations in the RPA binding regions of XPA on XPA function in NER. XPA binds through an N-terminal region to the middle subunit (RPA32) of the RPA heterotrimer and through a central region that overlaps with its damaged DNA binding region to the RPA70 subunit. In cell-free NER assays, an N-terminal deletion mutant of XPA showed loss of binding to RPA32 and reduced DNA repair activity, but it could still bind to UV-damaged DNA and RPA. In contrast, amino acid substitutions in the central region reduced incisions at the damaged site in the cell-free NER assay, and four of these mutants (K141A, T142A, K167A, and K179A) showed reduced binding to RPA70 but normal binding to damaged DNA. Furthermore, mutants that had one of the four aforementioned substitutions and an Nterminal deletion exhibited lower DNA incision activity and binding to RPA than XPA with only one of these substitutions or the deletion. Taken together, these results indicate that XPA interaction with both RPA32 and RPA70 is indispensable for NER reactions.

Back to Main Page1. Please note that only one statement of equally contributing authors is allowed (as well as one statement of joint supervision, if appropriate), so the two present statements have been amalgamated into one for now. If the two pairs of authors need to be diffentiated, a statement can be added to the author contributions section to explain. If you would like to do this, please provide some text to add.The meer fact that two different statements were made was because they were not the same. So they must not be amalgamated.I have added a line in the Author Contribution section.2. First para of main text, sentence beginning "In yeast", please check that "SlX4" is correct. Should it be SLX4?It should be Slx4 with lowercase L and lowercase X 3. Please check your article carefully, coordinate with any co-authors and enter all final edits clearly in the eproof, remembering to save frequently. Once corrections are submitted, we cannot routinely make further changes to the article. OK 4. Note that the eproof should be amended in only one browser window at any one time; otherwise changes will be overwritten.OK 5. Author surnames have been highlighted. Please check these carefully and adjust if the first name or surname is marked up incorrectly. Note that changes here will affect indexing of your article in public repositories such as PubMed. Also, carefully check the spelling and numbering of all author names and affiliations, and the corresponding email address(es). OK 6. You cannot alter accepted Supplementary Information files except for critical changes to scientific content. If you do resupply any files, please also provide a brief (but complete) list of changes. If these are not considered scientific changes, any altered Supplementary files will not be used, only the originally accepted version will be published. OK 7. Para beginning "Here, we unravel" -please check that PCNA has been expanded correctly at first mention. e.Proofing https://eproofing.springer.com/journals_v2/printpage.php?token... 1 of 64 22/04/2020, 15:41 OK 8. Results section, first para, please define YFP at first mention Yellow fluorescent protein (YFP) Figure 1b -please define IgCThis is a typo. It should be IgG in the figure and the following definition added to the legend:IgG: Immunoglobulin G 10. Figure 1c, bottom axes of plots -please check that the jump from 10 h to 12 h is correct (the other time points are in increments of 1 h). Please also check that the labels added to the right of the immunoblots in panel c are correct.All is correct Figure 1 caption -please define HA at first mentionHemagglutinin (HA) Figure 1 -Please check carefully that the caption for panel c is correct as edited to match the new figure.All is OK In the caption to Figure 2g,h -please differentiate (by inserting (g) and (h) what the two panels show.The caption is the same for g and h. The only difference is that the mutations in g are missense mutations while they are nonsense mutations in h. I have changed the caption to:Co-immunoprecipitation of en...

Topoisomerase 1 (Top1) is the intercellular target of camptothecins (CPTs). CPT blocks DNA religation in the Top1-DNA complex and induces Top1-attached nick DNA lesions. In this study, we demonstrate that excision repair cross complementing 1 protein-xeroderma pigmentosum group F (ERCC1-XPF) endonuclease and replication protein A (RPA) participate in the repair of Top1-attached nick DNA lesions together with other nucleotide excision repair (NER) factors. ERCC1-XPF shows nuclease activity in the presence of RPA on a 3'-phosphotyrosyl bond nick-containing DNA (Tyr-nick DNA) substrate, which mimics a Top1-attached nick DNA lesion. In addition, ERCC1-XPF and RPA form a DNA/protein complex on the nick DNA substrate in vitro, and co-localize in CPT-treated cells in vivo. Moreover, the DNA repair synthesis of Tyr-nick DNA lesions occurred in the presence of NER factors, including ERCC1-XPF, RPA, DNA polymerase delta, flap endonuclease 1 and DNA ligase 1. Therefore, some of the NER repair machinery might be an alternative repair pathway for Top1-attached nick DNA lesions. Clinically, these data provide insights into the potential of ERCC1 as a biomarker during CPT regimens.

XPG is a causative gene underlying the photosensitive disorder xeroderma pigmentosum group G (XP-G) and is involved in nucleotide excision repair. Here, we show that XPG knockdown represses epidermal growth factor (EGF)-induced FOS transcription at the level of transcription elongation with little effect on EGF signal transduction. XPG interacted with transcription elongation factors in concert with TFIIH, suggesting that the XPG-TFIIH complex serves as a transcription elongation factor. The XPG-TFIIH complex was recruited to promoter and coding regions of both EGF-induced (FOS) and housekeeping (EEF1A1) genes. Further, EGF-induced recruitment of RNA polymerase II and TFIIH to FOS was reduced by XPG knockdown. Importantly, EGF-induced FOS transcription was markedly lower in XP-G/Cockayne syndrome (CS) cells expressing truncated XPG than in control cells expressing wild-type (WT) XPG, with less significant decreases in XP-G cells with XPG nuclease domain mutations. In corroboration of this finding, both WT XPG and a missense XPG mutant from an XP-G patient were recruited to FOS upon EGF stimulation, but an XPG mutant mimicking a C-terminal truncation from an XP-G/CS patient was not. These results suggest that the XPG-TFIIH complex is involved in transcription elongation and that defects in this association may partly account for Cockayne syndrome in XP-G/CS patients.