Monitoring repair of UV-induced 6-4-photoproducts with a purified DDB2 protein complex.
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.
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America ; Department of Surgery, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
Matija
M
Dreze
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.
Anne S
AS
Calkins
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
Judit
J
Gálicza
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.
Daniel J
DJ
Echelman
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.
Mathew R
MR
Schnorenberg
Department of Surgery, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
Gillian L
GL
Fell
Division of Chemistry, Graduate School of Engineering Science, Osaka University, Osaka, Japan.
Shigenori
S
Iwai
Cutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America.
David E
DE
Fisher
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
David
D
Szüts
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America ; Department of Surgery, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.
J Dirk
JD
Iglehart
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America.
Jean-Bernard
JB
Lazaro
D002460:Cell Line; D004249:DNA Damage / Q000235:genetics / Q000502:physiology*; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D011740:Pyrimidine Dimers / Q000378:metabolism; D014466:Ultraviolet Rays
C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; D011740:Pyrimidine Dimers