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Monitoring Repair of UV-Induced 6-4-Photoproducts with a Purified DDB2 Protein Complex

Dreze, Matija and Calkins, Anne S. and Gálicza, Judit and Szüts, Dávid (2014) Monitoring Repair of UV-Induced 6-4-Photoproducts with a Purified DDB2 Protein Complex. PLOS ONE, 9 (1). e-0085896. ISSN 1932-6203

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Abstract

Because cells are constantly subjected to DNA damaging insults, DNA repair pathways are critical for genome integrity [1]. DNA damage recognition protein complexes (DRCs) recognize DNA damage and initiate DNA repair. The DNA-Damage Binding protein 2 (DDB2) complex is a DRC that initiates nucleotide excision repair (NER) of DNA damage caused by ultraviolet light (UV) [2]-[4]. Using a purified DDB2 DRC, we created a probe ("DDB2 proteo-probe") that hybridizes to nuclei of cells irradiated with UV and not to cells exposed to other genotoxins. The DDB2 proteo-probe recognized UV-irradiated DNA in classical laboratory assays, including cyto- and histo-chemistry, flow cytometry, and slot-blotting. When immobilized, the proteo-probe also bound soluble UV-irradiated DNA in ELISA-like and DNA pull-down assays. In vitro, the DDB2 proteo-probe preferentially bound 6-4-photoproducts [(6-4)PPs] rather than cyclobutane pyrimidine dimers (CPDs). We followed UV-damage repair by cyto-chemistry in cells fixed at different time after UV irradiation, using either the DDB2 proteo-probe or antibodies against CPDs, or (6-4)PPs. The signals obtained with the DDB2 proteo-probe and with the antibody against (6-4)PPs decreased in a nearly identical manner. Since (6-4)PPs are repaired only by nucleotide excision repair (NER), our results strongly suggest the DDB2 proteo-probe hybridizes to DNA containing (6-4)PPs and allows monitoring of their removal during NER. We discuss the general use of purified DRCs as probes, in lieu of antibodies, to recognize and monitor DNA damage and repair.

Item Type: Article
Additional Information: PMC3904869
Subjects: Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia
Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3011 Biochemistry / biokémia
R Medicine / orvostudomány > R1 Medicine (General) / orvostudomány általában
SWORD Depositor: MTMT SWORD
Depositing User: MTMT SWORD
Date Deposited: 08 May 2014 14:15
Last Modified: 09 May 2014 09:12
URI: http://real.mtak.hu/id/eprint/12712

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