Expression of the yeast PHR1 gene is induced by DNA-damaging agents.

Abstract

The PHR1 gene of Saccharomyces cerevisiae encodes a photolyase which repairs specifically and exclusively pyrimidine dimers, the most frequent lesions induced in DNA by far-UV radiation. We have asked whether expression of PHR1 is modulated in response to UV-induced DNA damage and to DNA-damaging agents that induce lesions structurally dissimilar to pyrimidine dimers. Using a PHR1-lacZ fusion gene in which expression of beta-galactosidase is regulated by PHR1 5' regulatory elements, we found that exposure of cells to 254-nm light, 4-nitroquinoline-N-oxide, methyl methanesulfonate, and N-methyl-N'-nitro-N-nitrosoguanidine induced synthesis of increased amounts of fusion protein. In contrast to these DNA-damaging agents, neither heat shock nor exposure to photoreactivating light elicited a response. Induction by far-UV radiation was evident both when the fusion gene was carried on a multicopy plasmid and when it replaced the endogenous chromosomal copy of PHR1, and it was accompanied by an increase in the steady-state concentration of PHR1-lacZ mRNA. Northern (RNA) blot analysis of PHR1 mRNA encoded by the chromosomal locus was consistent with either enhanced transcription of PHR1 after DNA damage or stabilization of the transcripts. Neither the intact PHR1 or RAD2 gene was required for induction. Comparison of the region of PHR1 implicated in regulation of its expression with other damage-inducible genes from yeast cells revealed a common conserved sequence that is present in the PHR1, RAD2, and RNR2 genes and is required for damage inducibility of the latter two genes. These sequences may constitute elements of a damage-responsive regulon in S. cerevisiae.

Role of UME6 in Transcriptional Regulation of a DNA Repair Gene in Saccharomyces cerevisiae

“…An important question is whether by removing UAS PHR1 from its native context, we altered its regulatory properties either by eliminating interactions between proteins bound to contiguous promoter elements or by fortuitously introducing a new regulatory site during construction of the reporter genes. To address this question, we introduced pGBS503 (UME6) into strain GBS231, in which the chromosomal copy of PHR1 has been replaced by an integrated PHR1-lacZ translational fusion (50). ␤-Galactosidase activities in two transformed isolates (GBS238 and GBS239) was compared to those seen in isogenic derivatives which had lost pGBS503 (GBS235, GBS236, and GBS237).…”

“…Plasmid structures were confirmed by restriction endonuclease digestion patterns and, where indicated, by DNA sequence analysis. Plasmids pBM1501, pPAL, pRS415, pRS416, pGBS116, pMAL-UME6, YEp24, pHY14-2, and pHY16-2 and the Carlson-Botstein Saccharomyces cerevisiae genomic library have been described previously (3,6,11,40,47,50,54,59). Plasmid pPRTCM was a gift from Howard Fried (University of North Carolina at Chapel Hill); it is a CEN plasmid carrying TRP1 and the TCM1-lacZ fusion from pTCMZ2 (15).…”

“…This enzyme catalyzes the lightdependent repair of cyclobutane-type pyrimidine dimers in DNA and stimulates "dark repair" of these lesions via a mechanism that requires an active nucleotide excision repair pathway (48). PHR1 transcription is induced by a variety of DNA-damaging agents, including 254-nm radiation, methylmethane sulfonate, 4-nitroquinoline oxide, nitrosoguanidine, and cisplatin (24,50), but is not induced by heat shock or photoreactivating light. Deletion and mutation analysis of PHR1 5Ј flanking sequences has identified three regulatory elements that contribute to basal-level and induced expression: a 22-bp palindrome (URS PHR1 ) that is the binding site for the damageresponsive repressor PRP, an upstream activation sequence (UAS PHR1 ) comprised of two DRC elements arranged as an interrupted molecular palindrome, and a novel essential sequence (UES PHR1 ) which antagonizes repressor binding or function (47,51).…”

Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

“…RAD4, RAD10, RAD16, RAI2]! RNR2 BNR3, DDR48, and PHRI DNA repair and DNA metabolism genes (15,21). (The underlined genes are known to be inducible by DNA-damaging agents.)…”

Rdp1, a Novel Zinc Finger Protein, Regulates the DNA Damage Response of rhp51+ from Schizosaccharomyces pombe

“…In particular, a large number of genes are induced in response to DNA damage and/or inhibition of DNA replication in Saccharomyces cerevisiae (22,49). These include RNR2 (which encodes a small subunit of ribonucleotide reductase [19,28]), RNR3 (which encodes a large subunit of ribonucleotide reductase [20]), CDC9 (which encodes DNA ligase [6]), and CDC17 (which encodes DNA polymerase I [21]), which are involved in DNA metabolism, and RAD2 (which encodes a DNA endonuclease required for nucleotide excision repair [NER] [48]), RAD7 (required for NER [48]), RAD18 (required for postreplication repair [34]), RAD23 (which encodes a uniquitin-like protein required for NER [41]), RAD51 (which encodes a RecA homolog required for double-strand break repair [7]), RAD54 (which encodes a putative DNA helicase required for double-strand break repair [13]), PHR1 (which encodes a photoreactivating enzyme [52]), and MAG (which encodes 3-methyladenine DNA glycosylase [12]), which are involved in DNA repair. However, the biological significance of the transcriptional induction of these genes has been uncovered only recently.…”