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

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.

Single Molecule Analysis of Laser Localized Interstrand Crosslinks

“…For example, a DNA reactive agent linked to one of the partners in azide/alkyne click chemistry would be applicable. Similarly, antibodies or specific binding proteins against DNA adducts (carcinogens, UV photoproducts) could also be employed ( Poirier, 1993 ; Dreze et al, 2014 ; McCready, 2014 ). Thus, we think it likely that the fiber strategies can be generalized to other DNA damaging agents, and could be used to address question regarding adduct distribution, repair, and replication fork encounters.…”

ERCC2Helicase Domain Mutations Confer Nucleotide Excision Repair Deficiency and Drive Cisplatin Sensitivity in Muscle-Invasive Bladder Cancer

“…The DDB2-FLAG-HA proteoprobe was purified from HeLa S3 cells as described previously (19). Briefly, cells were seeded on 24well PTFE printed slides (Electron Microscopy Sciences) at a density of 3,500 cells per well.…”

“…To test the impact of each ERCC2 mutation on NER capacity, we developed a novel fluorescence-based microscopy assay (19). The assay utilizes a purified DDB2 (damage-specific DNA-binding protein 2) proteoprobe that binds tightly and specifically to UV-induced (6,4)-photoproducts (Materials and Methods; ref.…”

Methods for the Detection of DNA Damage

“…3 The visualization of UV damages after local irradiation of Syrian hamster fibroblasts. DDB2 proteo-probe-binded (6-4)PPs that were induced preferentially by UVB and UVC but not by UVA suggest a possible use of this probe for the recognition and monitoring the repair of this certain type of UV lesions [84]. Fluorescence microscopy images of fibroblasts immunostained with mouse anti-CPD antibody and mouse anti-γH2AX antibody followed by Alexa Fluor 488-conjugated secondary antibodies.…”

“…The probe recognizes the UV-irradiated DNA in a number of assays, including cytochemistry, histochemistry, flow cytometry, slot-blotting, and DNA pull-down assays [84]. The probe recognizes the UV-irradiated DNA in a number of assays, including cytochemistry, histochemistry, flow cytometry, slot-blotting, and DNA pull-down assays [84].…”