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

Guerrero-Santoro, Jennifer; Kapetanaki, Maria G.; Hsieh, Ching Lung; Gorbachinsky, Ilya; Levine, Arthur S.; Rapić-Otrin, Vesna

doi:10.1158/0008-5472.can-07-6162

Cited by 114 publications

(99 citation statements)

References 46 publications

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“…chromatin relaxation, and enabling access to subsequent repair factors (14,(18)(19)(20). A current working model for damage recognition in global genomic repair is that UV photoproducts are first recognized by UV-DDB (18,21,22).…”

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confidence: 99%

“…A current working model for damage recognition in global genomic repair is that UV photoproducts are first recognized by UV-DDB (18,21,22). Stable binding of UV-DDB to sites of damage activates the ubiquitination activity of CRL4 DDB2 , which targets histones, primarily H2A, and enables nucleosome disassembly and subsequent recruitment of the XPC complex, which is also a ubiquitination substrate (18)(19)(20)(21)(22)(23)(24). Lesion handover between CRL4 DDB2 and XPC is thought to be achieved by the autoubiquitination of DDB2 at lysines in the intrinsically disordered N terminus of DDB2 (18,21).…”

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confidence: 99%

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

Ghodke

Wang

Hsieh

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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How human DNA repair proteins survey the genome for UVinduced 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.DNA damage recognition | single-molecule tracking | DNA tightrope | human nucleotide excision repair U nrepaired photoproducts in the genome arising from exposure to UV irradiation can be highly mutagenic and three pathways have evolved in mammalian cells to process these lesions, which include (i) global genomic repair, (ii) transcription-coupled repair, and (iii) translesion synthesis (1-5). During global genomic repair, cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts [(6-4) photoproducts] are repaired by the nucleotide excision repair (NER) pathway that recognizes and excises bulky helix distorting lesions in the genome (6, 7). The recognition of CPD lesions in UV-damaged chromatin is mediated by UV-damaged DNAbinding protein (UV-DDB), composed of the tightly associated heterodimer of damage-specific DNA binding protein (DDB) 1 (p127) and DDB2 (p48) (5,8). Following surveillance and CPD identification by UV-DDB, NER proceeds via lesion handover to XPC-hHR23B-centrin2 (XPC) followed by damage verification, helix opening and stabilizing of the repair intermediates, dual incision of the DNA in the context of the lesion, repair synthesis, and DNA ligation (7). In contrast to global genomic repair, transcription-coupled repair is initiated when CPD lesions in transcribed chromatin cause stalling of RNA polymerases (3). In mammalian NER, these two pathways converge after damage detection and are orchestrated by over 30 different gene products (9). Deficiencies in the molecular functions in seven of these NER proteins lead to various forms of the autosomal recessive disor...

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confidence: 99%

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confidence: 99%

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

Ghodke

Wang

Hsieh

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…In human and mouse CRL4 DDB2 , DDB2 acts as a DCAF to recognize specific DNA targets, and Cul4 then ubiquitylates nearby histones to open up nucleosomes for repair (37)(38)(39). However, in other cases, the target of the DCAF is also the substrate for Cul4.…”

Section: Discussionmentioning

confidence: 99%

CRL4-like Clr4 complex in Schizosaccharomyces pombe depends on an exposed surface of Dos1 for heterochromatin silencing

Kuscu

Zaratiegui

Kim

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Significance The CLRC complex is essential for heterochromatin formation in the yeast Schizosaccharomyces pombe. Its well-known role in placing methyl marks on histone H3 lysine 9 at heterochromatic loci is attributed to one of its components, cryptic loci regulator 4. However, it also contains an E3 ubiquitin ligase, a less understood activity of this complex. Here, we describe the organization of this seven-component complex and determine the crystal structure of delocalization of Swi6 1 (Dos1), a key subunit involved in targeting CLRC. We identify Dos2 as the central component of the complex and point of contact with Stc1, which bridges CLRC to the RNAi-induced transcriptional silencing complex, and show that heterochromatin formation is dependent on an exposed surface of Dos1. These results provide an unprecedented, high-resolution functional annotation of CLRC.

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“…The NER portion of this module includes CUL4B, an E3 ubiquitin ligase that binds to damaged DNA and ubiquitinates histone H2A, modifying the structure of chromatin and facilitating NER (15,27). NHEJ1 encodes a DNA repair protein that is essential for the nonhomologous end-joining pathway, which preferentially mediates repair of double-stranded breaks in DNA (16,28).…”

Section: Resultsmentioning

confidence: 99%

“…Platinum-DNA adducts formed upon exposure to platinum-based chemotherapies are thought to be primarily removed through the nucleotide excision repair pathway (NER). Using an in vitro assay that measures the number of AP sites on the DNA of oxaliplatin-treated cells, we found that siRNA silencing of CUL4B and NHEJ1, both with known roles in the repair of DNA damage via the NER (15,16), significantly increased the amount of DNA damage relative to oxaliplatin-treated control cells (Fig. 3A).…”

Section: Sumo1mentioning

confidence: 99%

Functional Genomics Reveals Diverse Cellular Processes That Modulate Tumor Cell Response to Oxaliplatin

Harradine

Kassner

Chow

et al. 2011

Molecular Cancer Research

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Oxaliplatin is widely used to treat colorectal cancer, as both adjuvant therapy for resected disease and palliative treatment of metastatic disease. However, a significant number of patients experience serious side effects, including prolonged neurotoxicity, from oxaliplatin treatment creating an urgent need for biomarkers of oxaliplatin response or resistance to direct therapy to those most likely to benefit. As a first step to improve selection of patients for oxaliplatin-based chemotherapy, we have conducted an in vitro cell-based small interfering RNA (siRNA) screen of 500 genes aimed at identifying genes whose loss of expression alters tumor cell response to oxaliplatin. The siRNA screen identified twenty-seven genes, which when silenced, significantly altered colon tumor cell line sensitivity to oxaliplatin. Silencing of a group of putative resistance genes increased the extent of oxaliplatin-mediated DNA damage and inhibited cell-cycle progression in oxaliplatin-treated cells. The activity of several signaling nodes, including AKT1 and MEK1, was also altered. We used cDNA transfection to overexpress two genes (LTBR and TMEM30A) that were identified in the siRNA screen as mediators of oxaliplatin sensitivity. In both instances, overexpression conferred resistance to oxaliplatin. In summary, this study identified numerous putative predictive biomarkers of response to oxaliplatin that should be studied further in patient specimens for potential clinical application. Diverse gene networks seem to influence tumor survival in response to DNA damage by oxaliplatin. Finally, those genes whose loss of expression (or function) is related to oxaliplatin sensitivity may be promising therapeutic targets to increase patient response to oxaliplatin. Mol Cancer Res; 9(2); 173-82. Ó2010 AACR.

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

Cited by 114 publications

References 46 publications

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

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

CRL4-like Clr4 complex in Schizosaccharomyces pombe depends on an exposed surface of Dos1 for heterochromatin silencing

Functional Genomics Reveals Diverse Cellular Processes That Modulate Tumor Cell Response to Oxaliplatin

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