ABSTRACT
How human DNA repair proteins survey the genome for UV-induced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). To understand this process, we performed single-molecule experiments, which revealed that the human UV-damaged DNA-binding protein (UV-DDB) performs a 3D search mechanism and displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps before forming long-lived, nonmotile UV-DDB dimers (DDB1-DDB2)2 at sites of damage. Analysis of the rates of dissociation for the transient binding molecules on both undamaged and damaged DNA show multiple dwell times over three orders of magnitude: 0.3-0.8, 8.1, and 113-126 s. These intermediate states are believed to represent discrete UV-DDB conformers on the trajectory to stable damage detection. DNA damage promoted the formation of highly stable dimers lasting for at least 15 min. The xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO, supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient. This study proposes that UV-DDB recognizes lesions via multiple kinetic intermediates, through a conformational proofreading mechanism.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/metabolism , DNA/radiation effects , Amino Acid Substitution , DNA/chemistry , DNA Damage , DNA-Binding Proteins/genetics , Humans , Kinetics , Microscopy, Fluorescence , Models, Molecular , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Osmolar Concentration , Protein Conformation , Protein Multimerization , Pyrimidine Dimers/metabolism , Pyrimidine Dimers/radiation effects , Quantum Dots , Ultraviolet Rays/adverse effects , Xeroderma Pigmentosum/genetics , Xeroderma Pigmentosum/metabolismABSTRACT
UV light-induced photoproducts are recognized and removed by the nucleotide-excision repair (NER) pathway. In humans, the UV-damaged DNA-binding protein (UV-DDB) is part of a ubiquitin E3 ligase complex (DDB1-CUL4A(DDB2)) that initiates NER by recognizing damaged chromatin with concomitant ubiquitination of core histones at the lesion. We report the X-ray crystal structure of the human UV-DDB in a complex with damaged DNA and show that the N-terminal domain of DDB2 makes critical contacts with two molecules of DNA, driving N-terminal-domain folding and promoting UV-DDB dimerization. The functional significance of the dimeric UV-DDB [(DDB1-DDB2)(2)], in a complex with damaged DNA, is validated by electron microscopy, atomic force microscopy, solution biophysical, and functional analyses. We propose that the binding of UV-damaged DNA results in conformational changes in the N-terminal domain of DDB2, inducing helical folding in the context of the bound DNA and inducing dimerization as a function of nucleotide binding. The temporal and spatial interplay between domain ordering and dimerization provides an elegant molecular rationale for the unprecedented binding affinities and selectivities exhibited by UV-DDB for UV-damaged DNA. Modeling the DDB1-CUL4A(DDB2) complex according to the dimeric UV-DDB-AP24 architecture results in a mechanistically consistent alignment of the E3 ligase bound to a nucleosome harboring damaged DNA. Our findings provide unique structural and conformational insights into the molecular architecture of the DDB1-CUL4A(DDB2) E3 ligase, with significant implications for the regulation and overall organization of the proteins responsible for initiation of NER in the context of chromatin and for the consequent maintenance of genomic integrity.
Subject(s)
DNA Damage , DNA Repair , DNA-Binding Proteins/chemistry , Chromatin/chemistry , Chromatin/genetics , Chromatin/metabolism , Crystallography, X-Ray , Cullin Proteins/chemistry , Cullin Proteins/metabolism , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA-Binding Proteins/metabolism , Humans , Microscopy, Atomic Force , Microscopy, Electron , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Nucleic Acid Conformation , Protein Binding , Protein Folding , Protein Multimerization , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
How the nucleotide excision repair (NER) machinery gains access to damaged chromatinized DNA templates and how the chromatin structure is modified to promote efficient repair of the non-transcribed genome remain poorly understood. The UV-damaged DNA-binding protein complex (UV-DDB, consisting of DDB1 and DDB2, the latter of which is mutated in xeroderma pigmentosum group E patients, is a substrate-recruiting module of the cullin 4B-based E3 ligase complex, DDB1-CUL4B(DDB2). We previously reported that the deficiency of UV-DDB E3 ligases in ubiquitinating histone H2A at UV-damaged DNA sites in the xeroderma pigmentosum group E cells contributes to the faulty NER in these skin cancer-prone patients. Here, we reveal the mechanism by which monoubiquitination of specific H2A lysine residues alters nucleosomal dynamics and subsequently initiates NER. We show that DDB1-CUL4B(DDB2) E3 ligase specifically binds to mononucleosomes assembled with human recombinant histone octamers and nucleosome-positioning DNA containing cyclobutane pyrimidine dimers or 6-4 photoproducts photolesions. We demonstrate functionally that ubiquitination of H2A Lys-119/Lys-120 is necessary for destabilization of nucleosomes and concomitant release of DDB1-CUL4B(DDB2) from photolesion-containing DNA. Nucleosomes in which these lysines are replaced with arginines are resistant to such structural changes, and arginine mutants prevent the eviction of H2A and dissociation of polyubiquitinated DDB2 from UV-damaged nucleosomes. The partial eviction of H3 from the nucleosomes is dependent on ubiquitinated H2A Lys-119/Lys-120. Our results provide mechanistic insight into how post-translational modification of H2A at the site of a photolesion initiates the repair process and directly affects the stability of the human genome.
Subject(s)
Histones/chemistry , Nucleosomes/chemistry , Ubiquitin-Protein Ligases/chemistry , Ubiquitinated Proteins/chemistry , Ultraviolet Rays , Amino Acid Substitution , Cell Line , Cullin Proteins/chemistry , DNA/chemistry , DNA/radiation effects , DNA Damage , DNA Repair , DNA-Binding Proteins/chemistry , Histones/genetics , Humans , Polyubiquitin/chemistry , Protein Binding , Protein Processing, Post-Translational , Pyrimidine Dimers/chemistry , UbiquitinationABSTRACT
By removing UV-induced lesions from DNA, the nucleotide excision repair (NER) pathway preserves the integrity of the genome. The UV-damaged DNA-binding (UV-DDB) protein complex is involved in the recognition of chromatin-embedded UV-damaged DNA, which is the least understood step of NER. UV-DDB consists of DDB1 and DDB2, and it is a component of the cullin 4A (CUL4A)-based ubiquitin ligase, DDB1-CUL4A(DDB2). We previously showed that DDB1-CUL4A(DDB2) ubiquitinates histone H2A at the sites of UV lesions in a DDB2-dependent manner. Mutations in DDB2 cause a cancer prone syndrome, xeroderma pigmentosum group E (XP-E). CUL4A and its paralog, cullin 4B (CUL4B), copurify with the UV-DDB complex, but it is unclear whether CUL4B has a role in NER as a separate E3 ubiquitin ligase. Here, we present evidence that CUL4A and CUL4B form two individual E3 ligases, DDB1-CUL4A(DDB2) and DDB1-CUL4B(DDB2). To investigate CUL4B's possible role in NER, we examined its subcellular localization in unirradiated and irradiated cells. CUL4B colocalizes with DDB2 at UV-damaged DNA sites. Furthermore, CUL4B binds to UV-damaged chromatin as a part of the DDB1-CUL4B(DDB2) E3 ligase in the presence of functional DDB2. In contrast to CUL4A, CUL4B is localized in the nucleus and facilitates the transfer of DDB1 into the nucleus independently of DDB2. Importantly, DDB1-CUL4B(DDB2) is more efficient than DDB1-CUL4A(DDB2) in monoubiquitinating histone H2A in vitro. Overall, this study suggests that DDB1-CUL4B(DDB2) E3 ligase may have a distinctive function in modifying the chromatin structure at the site of UV lesions to promote efficient NER.
Subject(s)
Chromatin/metabolism , Cullin Proteins/metabolism , DNA Damage , Histones/metabolism , Ubiquitin-Protein Ligases/metabolism , Ultraviolet Rays , Cell Nucleus/metabolism , Cells, Cultured , Chromatin/radiation effects , Cullin Proteins/genetics , DNA Repair/physiology , DNA-Binding Proteins/metabolism , HeLa Cells , Humans , Models, Biological , Multiprotein Complexes/metabolism , Protein Binding , Protein Transport/radiation effects , Tissue Distribution , Transfection , Ubiquitination/radiation effects , Ultraviolet Rays/adverse effectsABSTRACT
Xeroderma pigmentosum (XP) is a heritable human disorder characterized by defects in nucleotide excision repair (NER) and the development of skin cancer. Cells from XP group E (XP-E) patients have a defect in the UV-damaged DNA-binding protein complex (UV-DDB), involved in the damage recognition step of NER. UV-DDB comprises two subunits, products of the DDB1 and DDB2 genes, respectively. Mutations in the DDB2 gene account for the underlying defect in XP-E. The UV-DDB complex is a component of the newly identified cullin 4A-based ubiquitin E3 ligase, DDB1-CUL4A(DDB2). The E3 ubiquitin ligases recognize specific substrates and mediate their ubiquitination to regulate protein activity or target proteins for degradation by the proteasomal pathway. In this study, we have addressed the role of the UV-DDB-based E3 in NER and sought a physiological substrate. We demonstrate that monoubiquitinated histone H2A in native chromatin coimmunoprecipitates with the endogenous DDB1-CUL4A(DDB2) complex in response to UV irradiation. Further, mutations in DDB2 alter the formation and binding activity of the DDB1-CUL4A(DDB2) ligase, accompanied by impaired monoubiquitination of H2A after UV treatment of XP-E cells, compared with repair-proficient cells. This finding indicates that DDB2, as the substrate receptor of the DDB1-CUL4A-based ligase, specifically targets histone H2A for monoubiquitination in a photolesion-binding-dependent manner. Given that the loss of monoubiquitinated histone H2A at the sites of UV-damaged DNA is associated with decreased global genome repair in XP-E cells, this study suggests that histone modification, mediated by the XPE factor, facilitates the initiation of NER.
