Replication protein A binds to regmlatory elements in yeast DNA repair and DNA metabolism

Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in theMAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps ofDNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar doublestranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind theMAG URSi-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes. Alkylating agents covalently modify DNA to generate alkylated bases and are toxic, mutagenic, and carcinogenic. Some alkylated bases cause mutations because they miscode when replicated, and others cause cell death because they block DNA replication, preventing cells from proceeding properly through the cell cycle (1-3). If they are to avoid the mutagenic and cytotoxic effects of alkylating agents, cells must repair these alkylated bases before their DNA is replicated. Halting DNA replication when the genome is assaulted assures that DNA damage will be repaired before it has a chance to be encountered by the replication machinery. Saccharomyces cerevisiae, like Escherichia coli and mammalian cells, responds to DNA damage by inhibiting DNA replication and arresting cell cycle progression, thus preventing the replication of damaged chromosomes (3-6). These cells also respond by inducing the expression of numerous genes, and many of these genes are involved in DNA repair and DNA metabolism (7, 8). The regulation of DNA damage-inducible regulons has been well characterized in E. coli (8-11). The SOS response is induced when single-stranded DNA (ssDNA), generated in the locale of DNA damage, activates RecA protein to facilitate the cleavage and inactivation of the LexA transcriptional repressor, derepressing a set of genes involved in DNA repair, mutagenesis, recombination, and cell division (8). Over 40 genes are regulated by OxyR and SoxRS in response to oxidative stress (9, 10), and four genes are regulated by the Ada DNA methyltransferase in response to alkylating agents (11). Methyl transfer from a methylphosphotriester lesion to the Ada Cys-69 residue converts the Ada The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. protein into an efficient transcriptional activator for the induction of the following: (i) the Ada methyltransferase, which also repairs the potentially mutagenic 06-methylguanine and 04-methylthymine lesions; (ii) the AlkA 3-methyladenine (3MeAde) DNA glycosylase, which removes the replication blocking 3MeAde lesion plus various other lesions (11) and thus prevents cell death; and (iii) the AlkB and AidB proteins, whose precise functions are unknown. The * cerevisiae 3MeAde DNA glycosylase, the MAG gene product, displays homology to the E. coli AlkA 3MeAde DNA glycosylase (12). Like the alkA gene, MAG is induced when cells are exposed to alkylating agents (12-14), but unlike alkA, MAG induction is not specific for alkylating agents, does not involve DNA methyltransferase (14), and occurs upon exposure to several agents, including 4-nitroquinoline oxide, UV light, and y irradiation (15). Several S. cerevisiae genes involved in DNA repair, DNA metabolism, and protein modification are induced by DNA-damaging agents-namely, RAD2 (16), RAD7 (17), RAD18 (17), RAD23 (18), RAD51 (19), RAD54 (20), PHR1 (21), RAD6 (18), CDC9 (22), CDC17/POLl (7), UBI4 (23), RNR2 (24), and RNR3 (25). Like MAG, each gene can be induced by a variety of DNA-damaging agents, irrespective of whether the gene product actually helps the cell to deal with the damage produced by those agents. To explore eukaryotic gene expression in response to DNA damage, we identified theMAG regulatory elements (15). The MAG upstream region contains one activating sequence (UAS) and two repressing sequences (URS1 and URS2). Moreover, MAG URS1-like sequences are present in at least 11 other DNA repair and DNA metabolism genes-namely, MGT1, PHR1, RADI, RAD2, RAD4, RAD10, RAD16, RAD51, DDR48, RNR2, and RNR3 (15). Here we show that replication protein A (Rpa), a multifunctional protein believed to participate in the initiation and elongation steps ofDNA replication, in nucleotide excision repair, and in homologous recombination (26-28) is present in the protein-DNA complexes formed at theMAG URSl and URS2 elements. Our results suggest an additional role for Rpa in regulating the transcription of DNA repair and metabolism genes, and we propose a model that links the DNA damage-induced inhibition of replication with DNA damage-induced gene expression. MATERIALS AND METHODS Strains, Media, and Molecular Biology Methods. Yeast strain DBY747 (MATa his3-1 leu2-3,112 trpl-289 ura3-52) was grown at 30°C in either YPD medium or SD medium (29). Yeast transformation was carried out as described (30, 31). ,B-Galactosidase assays were as described (15, 32). A 24-bp oligonucleotide containing 20 bp of the MAG URS2 region (-180 to -161; see Table 1) was cloned between the Sal I and Abbreviations: UAS, upstream activating sequence; URS, upstream repressing sequence; MeMes, methyl methanesulfonate; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA. *To whom reprint requests should be addressed.