Vesna Rapić-Otrin

The Cullin 4B-Based UV-Damaged DNA-Binding Protein Ligase Binds to UV-Damaged Chromatin and Ubiquitinates Histone H2A

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 CUL4Bs 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.

HIV-1 Vpr Loads Uracil DNA Glycosylase-2 onto DCAF1, a Substrate Recognition Subunit of a Cullin 4A-RING E3 Ubiquitin Ligase for Proteasome-dependent Degradation

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, interacts with several host cellular proteins including uracil DNA glycosylase-2 (UNG2) and a cullin-RING E3 ubiquitin ligase assembly (CRL4DCAF1). The ligase is composed of cullin 4A (CUL4A), RING H2 finger protein (RBX1), DNA damage-binding protein 1 (DDB1), and a substrate recognition subunit, DDB1- and CUL4-associated factor 1 (DCAF1). Here we show that recombinant UNG2 specifically interacts with Vpr, but not with Vpx of simian immunodeficiency virus, forming a heterotrimeric complex with DCAF1 and Vpr in vitro as well as in vivo. Using reconstituted CRL4DCAF1 and CRL4DCAF1-Vpr E3 ubiquitin ligases in vitro reveals that UNG2 ubiquitination (ubiquitylation) is facilitated by Vpr. Co-expression of DCAF1 and Vpr causes down-regulation of UNG2 in a proteasome-dependent manner, with Vpr mutants that are defective in UNG2 or DCAF1 binding abrogating this effect. Taken together, our results show that the CRL4DCAF1 E3 ubiquitin ligase can be subverted by Vpr to target UNG2 for degradation.

Altered nucleotide misinsertion fidelity associated with polι‐dependent replication at the end of a DNA template

A hallmark of human DNA polymerase ι (polι) is the asymmetric fidelity of replication at template A and T when the enzyme extends primers annealed to a single‐stranded template. Here, we report on the efficiency and accuracy of polι‐dependent replication at a nick, a gap, the very end of a template and from a mispaired primer. Polι cannot initiate synthesis on a nicked DNA substrate, but fills short gaps efficiently. Surprisingly, polιs ability to blunt‐end a 1 bp recessed terminus is dependent upon the template nucleotide encountered and is highly erroneous. At template G, both C and T are inserted with roughly equal efficiency, whilst at template C, C and A are misinserted 8‐ and 3‐fold more often than the correct base, G. Using substrates containing mispaired primer termini, we show that polι can extend all 12 mispairs, but with differing efficiencies. Polι can also extend a tandem mispair, especially when it is located within a short gap. The enzymatic properties of polι appear consistent with that of a somatic hypermutase and suggest that polι may be one of the low‐fidelity DNA polymerases hypothesized to participate in the hypermutation of immunoglobulin variable genes in vivo.

Damaged DNA induced UV-damaged DNA-binding protein (UV-DDB) dimerization and its roles in chromatinized DNA repair

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-CUL4ADDB2) 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-CUL4ADDB2 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-CUL4ADDB2 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.

Monoubiquitinated Histone H2A Destabilizes Photolesion-containing Nucleosomes with Concomitant Release of UV-damaged DNA-binding Protein E3 Ligase

Background: The compaction of DNA into nucleosomes interferes with DNA repair. Results: Monoubiquitination of core histone H2A destabilizes nucleosomes containing UV-damaged DNA. Conclusion: Destabilized nucleosomes enable the release of the DNA damage-binding complex DDB1-CUL4BDDB2, which assists in histone ubiquitination. Significance: This mechanism explains how the ubiquitination of histone H2A, in addition to chromatin remodeling, promotes repair and facilitates genome stability. 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-CUL4BDDB2. 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-CUL4BDDB2 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-CUL4BDDB2 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.

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

Significance UV damage in genomic DNA is identified by the human UV-damaged DNA-binding protein (UV-DDB). Recognition of DNA damage by UV-DDB serves to initiate global genomic nucleotide excision repair (NER) in humans. Recent work has revealed that UV-DDB dimerizes at sites of damage. This study demonstrates that prior to stable damage recognition, UV-DDB interrogates DNA for damage via a 3D diffusion mechanism coupled to the formation of multiple transient intermediates. Stable binding at sites of damage is achieved by dimerization of UV-DDB. This study also analyzed a disease-causing mutant of UV-DDB, which was found to slide on DNA, while retaining the ability to dimerize on DNA. These results enhance our understanding of damage recognition in NER in humans. 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.

Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks.

During the DNA damage response (DDR), ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5), a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB) repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.

Co-localization of DNA Repair Proteins with UV-Induced DNA Damage in Locally Irradiated Cells

This chapter describes a technique in which indirect immunofluorescence is applied to visualize the process of nucleotide excision repair (NER) at the site of locally induced damage in DNA. UV-irradiation of cells through an isopore polycarbonate membrane filter generates cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PP) on a subnuclear area, which corresponds to the size of a pore on the membrane. Specific antibodies to CPD and 6-4PP define the damaged spot. The NER components co-localize at the damaged-DNA subnuclear spot, where the proteins are stained with the appropriate fluorescent antibodies. This relatively simple and affordable method facilitates the examination of the sequential assembly of NER proteins in the chromatin-embedded DNA photoproducts. The method also enhances the identification of repair auxiliary proteins and complexes, such as ubiquitin E3 ligases, involved in the initiation of NER on non-transcribed DNA.