Identification of New Genes Regulated by the Crt1 Transcription Factor, an Effector of the DNA Damage Checkpoint Pathway in Saccharomyces cerevisiae

Abstract

The Crt1 (RFX1) protein in Saccharomyces cerevisiae is an effector of the DNA damage checkpoint pathway. It recognizes a 13-bp cis-regulatory element in the 5′-untranslated region (5′-UTR) of the ribonucleotide reductase genes RNR2, RNR3, and RNR4; the HUG1 gene; and itself. We calculated the weight matrix representing the Crt1p binding site motif according to analysis of the 5′-UTR sequences of the genes that are under its regulation. We subsequently searched the 5′-UTR sequences of all the genes in the yeast genome for the occurrence of this motif. The motif was found in regulatory regions of 30 genes. A statistical analysis showed that it is unlikely that a random gene cluster contains the motif conserved as well as the Crt1p binding site. Analysis of microarray data provided supporting evidence for five putative Crt1p targets: FSH3, YLR345W, UBC5, NDE2, and NTH2. We used reverse transcription-PCR to compare the expression levels of these genes in wild-type and crt1Δ strains. Our results indicated that FSH3, YLR345W, and NTH2 are indeed under the regulation of Crt1p. Sequence analysis of the FSH3p indicated that this protein may be involved in folate metabolism either by carrying serine hydrolase activity required for the novel metabolic pathway involving dihydrofolate reductase (DHFR) or by directly interacting with the DHFR enzyme. We postulate that Crt1p may influence deoxyribonucleotide synthesis not only by regulating expression of the RNR genes but also by modulating DHFR activity. FSH3p shares significant sequence similarity with the product of the human tumor suppressor gene OVCA2. YLR345Wp and NTH2p are enzymes involved in the central metabolism under stress conditions. The Crt1 (RFX1) protein in Saccharomyces cerevisiae is an effector of the DNA damage checkpoint pathway. It recognizes a 13-bp cis-regulatory element in the 5′-untranslated region (5′-UTR) of the ribonucleotide reductase genes RNR2, RNR3, and RNR4; the HUG1 gene; and itself. We calculated the weight matrix representing the Crt1p binding site motif according to analysis of the 5′-UTR sequences of the genes that are under its regulation. We subsequently searched the 5′-UTR sequences of all the genes in the yeast genome for the occurrence of this motif. The motif was found in regulatory regions of 30 genes. A statistical analysis showed that it is unlikely that a random gene cluster contains the motif conserved as well as the Crt1p binding site. Analysis of microarray data provided supporting evidence for five putative Crt1p targets: FSH3, YLR345W, UBC5, NDE2, and NTH2. We used reverse transcription-PCR to compare the expression levels of these genes in wild-type and crt1Δ strains. Our results indicated that FSH3, YLR345W, and NTH2 are indeed under the regulation of Crt1p. Sequence analysis of the FSH3p indicated that this protein may be involved in folate metabolism either by carrying serine hydrolase activity required for the novel metabolic pathway involving dihydrofolate reductase (DHFR) or by directly interacting with the DHFR enzyme. We postulate that Crt1p may influence deoxyribonucleotide synthesis not only by regulating expression of the RNR genes but also by modulating DHFR activity. FSH3p shares significant sequence similarity with the product of the human tumor suppressor gene OVCA2. YLR345Wp and NTH2p are enzymes involved in the central metabolism under stress conditions. High fidelity of transmission of genetic information is crucial for the survival of living organisms. To assure faithful DNA replication, cells have evolved complex mechanisms that activate a multifaceted response to occasional DNA damage resulting from both environmental factors and cellular processes. In eukaryotes DNA damage activates evolutionarily conserved checkpoint pathways that induce apoptosis, arrest the cell cycle, and activate transcription of genes whose products are involved in repair processes. Signal transduction in these pathways proceeds through a kinase cascade involving products of the ATR, ATM, Chk1, and Chk2 genes in mammals and their yeast homologs MEC1, TEL1, CHK1, and RAD53 (1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2596) Google Scholar, 2Wahl G.M. Carr A.M. Nat. Cell Biol. 2001; 3: E277-E286Crossref PubMed Scopus (324) Google Scholar). The DNA damage response pathways and their evolutionary conservation in eukaryotic cells have been reviewed elsewhere (1Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Crossref PubMed Scopus (2596) Google Scholar, 2Wahl G.M. Carr A.M. Nat. Cell Biol. 2001; 3: E277-E286Crossref PubMed Scopus (324) Google Scholar). The product of the CRT1 gene in the yeast Saccharomyces cerevisiae is one of the effector proteins that take part in the DNA damage checkpoint pathway (3Huang M. Zhou Z. Elledge S.J. Cell. 1998; 94: 595-605Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). CRT1 encodes a DNA-binding protein that acts by recruiting Ssn6p and Tup1p, general repressors to the promoters of damage-inducible genes. In response to DNA damage Crt1p becomes phosphorylated via the Dun1p pathway downstream of the Rad53p checkpoint kinase. The hyperphosphorylated form of Crt1p does not bind DNA, preventing formation of a repressor complex, and leads to activation of damage-inducible genes. So far there are five experimentally verified targets of Crt1p action: three ribonucleotide reductase genes, RNR2, RNR3, and RNR4 (3Huang M. Zhou Z. Elledge S.J. Cell. 1998; 94: 595-605Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar), and the hydroxyurea/UV light/γ radiation-induced gene HUG1 (4Basrai M.A Velculescu V.E. Kinzler K.W. Hieter P. Mol. Cell Biol. 1999; 19: 7041-7049Crossref PubMed Scopus (91) Google Scholar). Crt1p also regulates expression of its own gene (3Huang M. Zhou Z. Elledge S.J. Cell. 1998; 94: 595-605Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). In the case of the RNR and CRT1 genes, Crt1p has been experimentally shown to recognize a 13-nucleotide-long cis-regulatory element (3Huang M. Zhou Z. Elledge S.J. Cell. 1998; 94: 595-605Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). The element resembles the mammalian X-box motif recognized by RFX transcription factors that take part in regulation of the major histocompatibility complex genes (5Reith W. Herrero-Sanchez C. Kobr M. Silacci P. Berte C. Barras E. Fey S. Mach B. PubMed Scopus Google Scholar, B. Kobr M. W. Mach B. Mol. Cell. Biol. PubMed Scopus Google Scholar). is not that the DNA-binding of Crt1p shares significant sequence similarity with the DNA-binding of RFX In the case of the HUG1 transcription binding has not been but the region of the gene contains 13-nucleotide-long the Crt1p binding (4Basrai M.A Velculescu V.E. Kinzler K.W. Hieter P. Mol. Cell Biol. 1999; 19: 7041-7049Crossref PubMed Scopus (91) Google Scholar). The Crt1p binding experimentally in the RNR and CRT1 genes, have been as or the binding of Crt1p and the of sequence conservation (3Huang M. Zhou Z. Elledge S.J. Cell. 1998; 94: 595-605Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). In RNR2, and one and one site have been contains one and (3Huang M. Zhou Z. Elledge S.J. Cell. 1998; 94: 595-605Abstract Full Text Full Text PDF PubMed Scopus (413) Google that binding response to the DNA damage the of Crt1p Crt1p from transcription experimentally verified Crt1p binding has been used to the binding site motif of the Crt1 transcription and its in regulatory regions of yeast genes. In this used a sequence motif to weight matrix representing the Crt1p binding site motif. The of yeast gene regulatory regions with the resulting motif a cluster of 30 putative Crt1p analysis of expression data to the of Crt1p We experimentally verified that three of YLR345W, and FSH3, are indeed by Crt1p. Analysis of data and the protein sequence of that the transduction pathway may deoxyribonucleotide synthesis not only the of ribonucleotide reductase activity but also by metabolism and The that and NTH2 are by Crt1p the regulatory DNA damage response and central of 5′-untranslated regions used dihydrofolate for motif reverse one occurrence of all in the S. cerevisiae genome from the Saccharomyces M. M. S. 2000; PubMed Scopus Google Scholar). M. C. S. M. C. S. and sequences of Crt1p binding from (3Huang M. Zhou Z. Elledge S.J. Cell. 1998; 94: 595-605Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). expression analysis microarray data of M. S. Elledge S.J. Mol. Biol. Cell. 2001; PubMed Scopus Google from the and the data of P. G.M. Mol. Cell. Biol. 2000; PubMed Scopus Google from of E. A.M. M. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google from site The for microarray data to this was by the yeast S. C. P. PubMed Scopus Google Scholar). The P. P. C. 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Kinzler K.W. Hieter P. Mol. Cell Biol. 1999; 19: 7041-7049Crossref PubMed Scopus (91) Google Scholar). analysis the sequences a motif that is conserved the Crt1p cis-regulatory element and an of the of its occurrence in random to the of the motif. We used to from to in the 5′-UTR sequences of RNR2, RNR3, and HUG1 genes. 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M. 2001; PubMed Google and the putative Crt1p binding shown by the in the of in the regulatory site is by genes and in the are in genes the in by The of the HUG1 gene is that a putative Crt1p binding site in the regulatory region of this gene is also from the gene downstream of HUG1 of gene does not the repressor complex (4Basrai M.A Velculescu V.E. Kinzler K.W. Hieter P. Mol. Cell Biol. 1999; 19: 7041-7049Crossref PubMed Scopus (91) Google are putative Crt1p targets that to of the genes experimentally shown to be under Crt1p regulation. The site the regulatory region of NTH2 is to the putative site of the HUG1 contains a site to the experimentally verified binding site the 5′-UTR of the RNR4 and genes. 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PubMed Scopus Google Scholar). and NTH2 enzymes involved in the regulation of central metabolism under stress conditions. of these enzymes by Crt1p may the cell to its central metabolism to the of DNA repair and survival under the of used the 5′-UTR sequences of the regulatory targets of the Crt1 transcription to the weight matrix representing its binding site sequence motif. this weight a significant cluster of 30 putative Crt1p targets was microarray data provided supporting evidence that five genes, FSH3, UBC5, NDE2, and YLR345W, are to regulation by Crt1p. We showed by of levels in crt1Δ and wild-type of S. cerevisiae that FSH3, YLR345W, and NTH2 are indeed under the of Crt1p. It is also that and NTH2 are under the of the stress response element regulatory sequence an of the the DNA damage and general stress response Sequence analysis of indicated that this protein may be involved in folate metabolism either by carrying serine hydrolase activity required for a novel metabolic pathway involving DHFR or by directly interacting with this is Crt1p regulates synthesis of the not only the of ribonucleotide but also the of and a for of a of eukaryotic proteins involving the human tumor suppressor gene OVCA2. YLR345Wp shares significant sequence similarity with enzymes for the metabolism of an for the regulation of and NTH2 encodes involved in the regulation of We postulate that both NTH2p and YLR345Wp are for of central metabolism the stress The that Crt1p regulates the ribonucleotide reductase genes that this effector of the DNA damage pathway is for the of the of the cell for DNA repair processes. The putative of the three genes this the influence of the DNA damage checkpoint pathway folate and to the complex of the of for DNA damage response in eukaryotic We are to for the of the DNA damage checkpoint pathway to and for of the We are to for We are also to for of the