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Vesna Rapic-Otrin, Ph.D.
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Assistant Professor
2.6 HCCLB
5117 Centre Avenue
Pittsburgh, Pennsylvania 15213
Phone: (412) 623-5962
Fax: (412) 623-7761
E-mail: vesnaro@pitt.edu
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Research
To preserve the integrity of the genome, cells have developed various sophisticated mechanisms for repairing damaged DNA. The major DNA repair process that removes helixdistorting lesions from DNA, including UV-induced cyclobutane pyrimidine dimers (CPD) and 6,4 PhotoProducts (6,4-PP) is the nucleotide excision repair (NER) pathway. However, in eukaryotic cells, NER operates on chromatin-embedded DNA substrates and DNA folding with histone proteins into chromatin poses structural constraints likely to challenge detection and repair of DNA lesions. Only recently there has been an emphasis on the relationship of chromatin to NER. It is a general notion that this early step requires chromatin-modifying or remodeling activity as a means to overcome the chromatin barrier. A current model, which delineates how repair of DNA lesions operates within chromatin, proposes that, in a first step, chromatin organization is transiently disrupted to facilitate access of the repair machinery to DNA lesions. The model implies that specific NER factors, presumably damage recognition proteins, direct chromatin modifiers or chromatin remodeling activities to the correct location in the vicinity of the CPD and (6,4-PP). The hypothesis is supported by our recent publication, where we demonstrated connection between the UV-damaged DNA-binding complex (UVDDB) and ubiquitylation of the histones in cellular response to UV-irradiation.
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. We investigate the role of the UV-DDB-based E3 in initiation of NER and sought a physiological substrate. We found 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.
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Selected Publications
- Guerrero-Santoro J, Kapetanaki MG, Hsieh CL, Gorbachinsky I, Levine AS, Rapić-Otrin V. 2008. "The cullin 4B-based UV-damaged DNA-binding protein ligase binds to UV-damaged chromatin and ubiquitinates histone H2A" Cancer Res. 68:5014-22. | Abstract
- Kapetanaki MG, Guerrero-Santoro J, Bisi DC, Hsieh CL, Rapić-Otrin V, Levine AS. 2006. "The DDB1-CUL4ADDB2 ubiquitin ligase is deficient in xeroderma pigmentosum group E and targets histone H2A at UV-damaged DNA sites" Proc Natl Acad Sci USA. 103:2588-93. | Abstract
- Rapić-Otrin V, Navazza V, Nardo T, Botta E, McLenigan M, Bisi DC, Levine AS, Stefanini M. 2003. "True XP group E patients have a defective UV-damaged DNA binding protein complex and mutations in DDB2 which reveal the functional domains of its p48 product" Hum Mol Genet. 12:1507-22. | Abstract
- Rapić-Otrin V, McLenigan MP, Bisi DC, Gonzalez M, Levine AS. 2002. "Sequential binding of UV DNA damage binding factor and degradation of the p48 subunit as early events after UV irradiation" Nucleic Acids Res. 30:2588-98. | Abstract
- Frank EG, Tissier A, McDonald JP, Rapić-Otrin V, Zeng X, Gearhart PJ, Woodgate R. 2001. "Altered nucleotide misinsertion fidelity associated with poliota-dependent replication at the end of a DNA template" EMBO J. 20:2914-22. | Abstract
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