Monitoring repair of UV-induced 6-4-photoproducts with a purified DDB2 protein complex

Matija Dreze, Anne S. Calkins, Judit Gálicza, Daniel J. Echelman, Mathew R. Schnorenberg, Gillian L. Fell, Shigenori Iwai, David E. Fisher, D. Szüts, J. Dirk Iglehart, Jean Bernard Lazaro

Research output: Contribution to journalArticle

6 Citations (Scopus)

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.

Original languageEnglish
Article numbere85896
JournalPLoS One
Volume9
Issue number1
DOIs
Publication statusPublished - Jan 28 2014

Fingerprint

DNA-Binding Proteins
Ultraviolet Rays
DNA damage
DNA Damage
ultraviolet radiation
binding proteins
Carrier Proteins
Repair
probes (equipment)
Monitoring
monitoring
DNA
DNA repair
DNA Repair
Proteins
proteins
Pyrimidine Dimers
chemistry
pyrimidines
antibodies

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Dreze, M., Calkins, A. S., Gálicza, J., Echelman, D. J., Schnorenberg, M. R., Fell, G. L., ... Lazaro, J. B. (2014). Monitoring repair of UV-induced 6-4-photoproducts with a purified DDB2 protein complex. PLoS One, 9(1), [e85896]. https://doi.org/10.1371/journal.pone.0085896

Monitoring repair of UV-induced 6-4-photoproducts with a purified DDB2 protein complex. / Dreze, Matija; Calkins, Anne S.; Gálicza, Judit; Echelman, Daniel J.; Schnorenberg, Mathew R.; Fell, Gillian L.; Iwai, Shigenori; Fisher, David E.; Szüts, D.; Iglehart, J. Dirk; Lazaro, Jean Bernard.

In: PLoS One, Vol. 9, No. 1, e85896, 28.01.2014.

Research output: Contribution to journalArticle

Dreze, M, Calkins, AS, Gálicza, J, Echelman, DJ, Schnorenberg, MR, Fell, GL, Iwai, S, Fisher, DE, Szüts, D, Iglehart, JD & Lazaro, JB 2014, 'Monitoring repair of UV-induced 6-4-photoproducts with a purified DDB2 protein complex', PLoS One, vol. 9, no. 1, e85896. https://doi.org/10.1371/journal.pone.0085896
Dreze M, Calkins AS, Gálicza J, Echelman DJ, Schnorenberg MR, Fell GL et al. Monitoring repair of UV-induced 6-4-photoproducts with a purified DDB2 protein complex. PLoS One. 2014 Jan 28;9(1). e85896. https://doi.org/10.1371/journal.pone.0085896
Dreze, Matija ; Calkins, Anne S. ; Gálicza, Judit ; Echelman, Daniel J. ; Schnorenberg, Mathew R. ; Fell, Gillian L. ; Iwai, Shigenori ; Fisher, David E. ; Szüts, D. ; Iglehart, J. Dirk ; Lazaro, Jean Bernard. / Monitoring repair of UV-induced 6-4-photoproducts with a purified DDB2 protein complex. In: PLoS One. 2014 ; Vol. 9, No. 1.
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