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The cullin–RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A–RBX1–DDB1–DDB2 complex (CRL4A DDB2 ) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A DBB2 is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A DDB2 and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN. Much of the intracellular protein degradation in eukaryotes is controlled by cullin–RING ubiquitin ligases (CRLs), a vast class of enzymes which are regulated by the COP9 signalosome (CSN); structural characterization of CSN–N8CRL4A complexes by cryo-electron microscopy reveals an induced-fit mechanism of CSN activation triggered only by catalytically activated CRLs without bound substrate, explaining how CSN acts as a global regulator of CRLs. Control of intracellular protein degradation Much of the intracellular protein degradation in eukaryotes is controlled by cullin–RING ubiquitin ligases (CRLs). The structure of these enzymes and their substrates vary greatly, yet all are regulated by a single complex — the COP9 signalosome (CSN). What enables CSN to be a master regulator of diverse CRLs? Nicolas Thomä and colleagues present biochemical data and cryo-electron microscopy of CSN–CRL4 complexes revealing an induced-fit mechanism that activates CSN only in the presence of a catalytically activated CRL not bound to a substrate. The authors identify both unique and less-specific CSN–CRL contacts.

In mammalian global genomic nucleotide excision repair, UV-DDB plays a central role in recognizing DNA lesions, such as 6-4 photoproducts and cyclobutane pyrimidine dimers, within chromatin. In the present study, we perform cryo-electron microscopy analyses coupled with chromatin-immunoprecipitation to reveal that the cellular UV-DDB binds to UV-damaged DNA lesions in a chromatin unit, the nucleosome, at a position approximately 20 base-pairs from the nucleosomal dyad in human cells. An alternative analysis of the in vitro reconstituted UV-DDB-cyclobutane pyrimidine dimer nucleosome structure demonstrates that the DDB2 subunit of UV-DDB specifically recognizes the cyclobutane pyrimidine dimer lesion at this position on the nucleosome. We also determine the structures of UV-DDB bound to DNA lesions at other positions in purified cellular human nucleosomes. These cellular and reconstituted UV-DDB-nucleosome complex structures provide important evidence for understanding the mechanism by which UV lesions in chromatin are recognized and repaired in mammalian cells. UV-DDB is a protein that plays a key role in recognizing DNA lesions. Here, the authors determine the cryo-EM structure of UV-DDB bound to UV-damaged chromatin in human cells, identifying a nucleosome binding site.

Nucleotide excision repair (NER) is a versatile DNA repair pathway, which can remove an extremely broad range of base lesions from the genome. In mammalian global genomic NER, the XPC protein complex initiates the repair reaction by recognizing sites of DNA damage, and this depends on detection of disrupted/destabilized base pairs within the DNA duplex. A model has been proposed that XPC first interacts with unpaired bases and then the XPD ATPase/helicase in concert with XPA verifies the presence of a relevant lesion by scanning a DNA strand in 5′-3′ direction. Such multi-step strategy for damage recognition would contribute to achieve both versatility and accuracy of the NER system at substantially high levels. In addition, recognition of ultraviolet light (UV)-induced DNA photolesions is facilitated by the UV-damaged DNA-binding protein complex (UV-DDB), which not only promotes recruitment of XPC to the damage sites, but also may contribute to remodeling of chromatin structures such that the DNA lesions gain access to XPC and the following repair proteins. Even in the absence of UV-DDB, however, certain types of histone modifications and/or chromatin remodeling could occur, which eventually enable XPC to find sites with DNA lesions. Exploration of novel factors involved in regulation of the DNA damage recognition process is now ongoing.

The DNA nucleotide excision repair (NER) system is our major defense against carcinogenesis. Defects in NER are associated with several human genetic disorders including xeroderma pigmentosum (XP), which is characterized by a marked predisposition to skin cancer. For initiation of the repair reaction at the genome-wide level, a complex containing one of the gene products involved in XP, the XPC protein, must bind to the damaged DNA site. The UV-damaged DNA-binding protein (UV-DDB), which is impaired in XP group E patients, has also been implicated in damage recognition in global genomic NER, but its precise functions and its relationship to the XPC complex have not been elucidated. However, the recent discovery of the association of UV-DDB with a cullin-based ubiquitin ligase has functionally linked the two damage recognition factors and shed light on novel mechanistic and regulatory aspects of global genomic NER. This article summarizes our current knowledge of the properties of the XPC complex and UV-DDB and discusses possible roles for ubiquitylation in the molecular mechanisms that underlie the efficient recognition and repair of DNA damage, particularly that induced by ultraviolet light irradiation, in preventing damage-induced mutagenesis as well as carcinogenesis.

The ubiquitin–proteasome system (UPS) plays crucial roles in regulation of various biological processes, including DNA repair. In mammalian global genome nucleotide excision repair (GG-NER), activation of the DDB2-associated ubiquitin ligase upon UV-induced DNA damage is necessary for efficient recognition of lesions. To date, however, the precise roles of UPS in GG-NER remain incompletely understood. Here, we show that the proteasome subunit PSMD14 and the UPS shuttle factor RAD23B can be recruited to sites with UV-induced photolesions even in the absence of XPC, suggesting that proteolysis occurs at DNA damage sites. Unexpectedly, sustained inhibition of proteasome activity results in aggregation of PSMD14 (presumably with other proteasome components) at the periphery of nucleoli, by which DDB2 is immobilized and sequestered from its lesion recognition functions. Although depletion of PSMD14 alleviates such DDB2 immobilization induced by proteasome inhibitors, recruitment of DDB2 to DNA damage sites is then severely compromised in the absence of PSMD14. Because all of these proteasome dysfunctions selectively impair removal of cyclobutane pyrimidine dimers, but not (6–4) photoproducts, our results indicate that the functional integrity of the proteasome is essential for the DDB2-mediated lesion recognition sub-pathway, but not for GG-NER initiated through direct lesion recognition by XPC.

Ribonucleoside triphosphates are often incorporated into genomic DNA during DNA replication. The accumulation of unrepaired ribonucleotides is associated with genomic instability, which is mediated by DNA topoisomerase 1 (Top1) processing of embedded ribonucleotides. The cleavage initiated by Top1 at the site of a ribonucleotide leads to the formation of a Top1-DNA cleavage complex (Top1cc), occasionally resulting in a DNA double-strand break (DSB). In humans, tyrosyl-DNA phosphodiesterases (TDPs) are essential repair enzymes that resolve the trapped Top1cc followed by downstream repair factors. However, there is limited cellular evidence of the involvement of TDPs in the processing of incorporated ribonucleotides in mammals. We assessed the role of TDPs in mutagenesis induced by a single ribonucleotide embedded into DNA. A supF shuttle vector site-specifically containing a single riboguanosine (rG) was introduced into the human lymphoblastoid TK6 cell line and its TDP1 -, TDP2 -, and TDP1 / TDP2 -deficient derivatives. TDP1 and TDP2 insufficiency remarkably decreased the mutant frequency caused by an embedded rG. The ratio of large deletion mutations induced by rG was also substantially lower in TDP1 / TDP2 -deficient cells than wild-type cells. Furthermore, the disruption of TDPs reduced the length of rG-mediated large deletion mutations. The recovery ratio of the propagated plasmid was also increased in TDP1 / TDP2 -deficient cells after the transfection of the shuttle vector containing rG. The results suggest that TDPs-mediated ribonucleotide processing cascade leads to unfavorable consequences, whereas in the absence of these repair factors, a more error-free processing pathway might function to suppress the ribonucleotide-induced mutagenesis. Furthermore, base substitution mutations at sites outside the position of rG were detected in the supF gene via a TDPs-independent mechanism. Overall, we provide new insights into the mechanism of mutagenesis induced by an embedded ribonucleotide in mammalian cells, which may lead to the fatal phenotype in the ribonucleotide excision repair deficiency.

How the fate (folding versus degradation) of glycoproteins is determined in the endoplasmic reticulum (ER) is an intriguing question. Monoglucosylated glycoproteins are recognized by lectin chaperones to facilitate their folding, whereas glycoproteins exposing well-trimmed mannoses are subjected to glycoprotein ER-associated degradation (gpERAD); we have elucidated how mannoses are sequentially trimmed by EDEM family members (George et al., 2020; 2021 eLife). Although reglucosylation by UGGT was previously reported to have no effect on substrate degradation, here we directly tested this notion using cells with genetically disrupted UGGT1/2. Strikingly, the results showed that UGGT1 delayed the degradation of misfolded substrates and unstable glycoproteins including ATF6α. An experiment with a point mutant of UGGT1 indicated that the glucosylation activity of UGGT1 was required for the inhibition of early glycoprotein degradation. These and overexpression-based competition experiments suggested that the fate of glycoproteins is determined by a tug-of-war between structure formation by UGGT1 and degradation by EDEMs. We further demonstrated the physiological importance of UGGT1, since ATF6α cannot function properly without UGGT1. Thus, our work strongly suggests that UGGT1 is a central factor in ER protein quality control via the regulation of both glycoprotein folding and degradation.

UV-DDB, an initiation factor for the nucleotide excision repair pathway, recognizes 6–4PP lesions through a base flipping mechanism. As genomic DNA is almost entirely accommodated within nucleosomes, the flipping of the 6–4PP bases is supposed to be extremely difficult if the lesion occurs in a nucleosome, especially on the strand directly contacting the histone surface. Here we report that UV-DDB binds efficiently to nucleosomal 6–4PPs that are rotationally positioned on the solvent accessible or occluded surface. We determined the crystal structures of nucleosomes containing 6–4PPs in these rotational positions and found that the 6–4PP DNA regions were flexibly disordered, especially in the strand exposed to the solvent. This characteristic of 6–4PP may facilitate UV-DDB binding to the damaged nucleosome. We present the first atomic-resolution pictures of the detrimental DNA cross-links of neighboring pyrimidine bases within the nucleosome and provide the mechanistic framework for lesion recognition by UV-DDB in chromatin.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.

Fanconi anemia (FA) is a rare genetic disease characterized by the deficiency of the cellular response and repair pathway for DNA interstrand crosslink (ICL) damage. Although recent studies have revealed the detailed molecular functions of FA proteins encoded by 22 genes, the mechanism of occurrence of endogenous ICLs in the human body remains poorly understood. In this short review, we summarize the potential endogenous sources of ICLs counteracted by FA proteins, and provide perspectives on the unanswered questions regarding FA.