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.
Highlights
Response to DNA damage caused by genotoxic stress involves recognition of the damage and subsequent repair
Specific DNA damage recognition protein complexes (DRCs) recognize and bind the various lesions found in DNA to initiate their cognate DNA repair pathway
We found the DNA Damage Binding protein 2 complex (DDB2) proteo-probe hybridized to the nuclei of cells irradiated with ultraviolet light (UV)-B, but not UV-A (Figure S2)
Summary
Response to DNA damage caused by genotoxic stress involves recognition of the damage and subsequent repair. Specific DNA damage recognition protein complexes (DRCs) recognize and bind the various lesions found in DNA to initiate their cognate DNA repair pathway. Upon reaching DNA, UV light predominantly causes intra-strand crosslinks of two adjacent pyrimidines, causing cyclobutane pyrimidine dimers (CPDs) and 6-4-photoproducts [(6-4)PPs] [6,7]. Both types of lesions are repaired by the nucleotide excision repair pathway (NER), albeit on different time scales. Recognition of UV damaged DNA by the DNA Damage Binding protein 2 complex (DDB2) is necessary for the timely completion of global genome repair (GGR) of UV lesions by NER in vivo [8,9,10,11]. A crystal structure of DDB2 bound to (6-4)PPs or CPDs have been resolved [15,16]
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