title: Preferential binding of yeast Rad4.Rad23 complex to damaged DNA.
abstract: The yeast Rad4 and Rad23 proteins form a complex that is involved in nucleotide excision repair (NER). Their function in this process is not known yet, but genetic data suggest that they act in an early step in NER. We have purified an epitope-tagged Rad4.Rad23 (tRad4. Rad23) complex from yeast cells, using a clone overproducing Rad4 with a hemagglutinin-tag at its C terminus. tRad4.Rad23 complex purified by both conventional and immuno-affinity chromatography complements the in vitro repair defect of rad4 and rad23 mutant extracts, demonstrating that these proteins are functional in NER. Using electrophoretic mobility shift assays, we show preferential binding of the tRad4.Rad23 complex to damaged DNA in vitro. UV-irradiated, as well as N-acetoxy-2-(acetylamino)fluorene-treated DNA, is efficiently bound by the protein complex. These data suggest that Rad4.Rad23 interacts with DNA damage during NER and may play a role in recognition of the damage.
authors.affiliation: MGC Department of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, P. O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: L E
authors.initials: LE
authors.lastname: Jansen
authors.forename: R A
authors.initials: RA
authors.lastname: Verhage
authors.forename: J
authors.initials: J
authors.lastname: Brouwer
mesh_terms: D000099:Acetoxyacetylaminofluorene / Q000494:pharmacology; D001483:Base Sequence; D004247:DNA / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D011503:Transglutaminases; D014466:Ultraviolet Rays
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C490846:RAD23 protein, S cerevisiae; C082463:RAD4 protein, S pombe; C490845:RHP23 protein, S pombe; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D000099:Acetoxyacetylaminofluorene; D004247:DNA; D011503:Transglutaminases