A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1.
The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. We report here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 upstream repression sequence, that together regulate induction of PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. A prominent feature of the foot-printed region is a 22-base-pair palindrome. Deletion of the PHR1 upstream repression sequence increased the basal level expression of PHR1 in vivo and decreased induction after exposure of cells to UV radiation or methyl methanesulfonate, whereas insertion of the PRP binding site between the CYC1 upstream activation sequence and "TATA" sequence reduced basal level expression and conferred damage responsiveness upon a reporter gene. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription.
Sebastian
J
J
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599-7260.
Sancar
G B
GB
D001483:Base Sequence; D001665:Binding Sites; D004249:DNA Damage; D004260:DNA Repair / Q000235:genetics*; D004271:DNA, Fungal / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000528:radiation effects; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D008741:Methyl Methanesulfonate; D001616:beta-Galactosidase; D004255:Deoxyribodipyrimidine Photo-Lyase