Requirement of DNA Polymerase Activity of Yeast Rad30 Protein for Its Biological Function

The RAD30 gene of Saccharomyces cerevisiae encodes a DNA polymerase, Polη. The Rad30 protein shares homology with the yeast Rev1 and the Escherichia coli DinB and UmuC proteins. Although these proteins contain several highly conserved motifs, only Rad30 has been shown to possess a DNA polymerase activity. To determine whether the DNA polymerase activity of Rad30 was essential for its biological function, we made a mutation in the highly conserved SIDE sequence in Rad30, in which the aspartate and glutamate residues have each been changed to alanine. The mutant Rad30 protein lacks the DNA polymerase activity, and the mutant gene does not complement the rad30Δ mutation. These findings indicate that DNA polymerase activity is indispensable for the biological function of RAD30. The RAD30 gene of Saccharomyces cerevisiae encodes a DNA polymerase, Polη. The Rad30 protein shares homology with the yeast Rev1 and the Escherichia coli DinB and UmuC proteins. Although these proteins contain several highly conserved motifs, only Rad30 has been shown to possess a DNA polymerase activity. To determine whether the DNA polymerase activity of Rad30 was essential for its biological function, we made a mutation in the highly conserved SIDE sequence in Rad30, in which the aspartate and glutamate residues have each been changed to alanine. The mutant Rad30 protein lacks the DNA polymerase activity, and the mutant gene does not complement the rad30Δ mutation. These findings indicate that DNA polymerase activity is indispensable for the biological function of RAD30. UV-induced DNA damage presents a block to the DNA replication machinery. To maintain the continuity of the DNA during replication, UV lesions encountered by the replication machinery are circumvented by both error-free and error-prone means. In the yeast Saccharomyces cerevisiae, genes in the RAD6 epistasis group function in the replication of DNA-containing lesions generated by UV light and by other DNA damaging agents. Mutations in the RAD6 andRAD18 genes confer extreme sensitivity to UV light, and these mutants are defective in postreplicative bypass of UV-damaged DNA and in UV-induced mutagenesis (1Prakash S. Sung P. Prakash L. Annu. Rev. Genet. 1993; 27: 33-70Crossref PubMed Scopus (256) Google Scholar). Rad6, a ubiquitin-conjugating enzyme, exists in vivo in a complex with Rad18, a DNA-binding protein (2Bailly V. Lamb J. Sung P. Prakash S. Prakash L. Genes Dev. 1994; 8: 811-820Crossref PubMed Scopus (277) Google Scholar, 3Bailly V. Lauder S. Prakash S. Prakash L. J. Biol. Chem. 1997; 272: 23360-23365Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). How Rad6-Rad18 protein-dependent ubiquitination promotes error-free and mutagenic postreplicative bypass is not known.Of the genes in the RAD6 epistasis group,REV1, REV3, and REV7 are required for mutagenic bypass of UV damage, and yeast lacking any of these genes is nonmutable by UV light (1Prakash S. Sung P. Prakash L. Annu. Rev. Genet. 1993; 27: 33-70Crossref PubMed Scopus (256) Google Scholar). The Rev3 and Rev7 proteins form DNA polymerase ζ, which shows limited ability to bypass acis-syn thymine-thymine dimer (4Nelson J.R. Lawrence C.W. Hinkle D.C. Science. 1996; 272: 1646-1649Crossref PubMed Scopus (595) Google Scholar). Rev1 is a deoxycytidyl transferase that can incorporate a dCMP residue opposite an abasic site (5Nelson J.R. Lawrence C.W. Hinkle D.C. Nature. 1996; 382: 729-731Crossref PubMed Scopus (505) Google Scholar). The RAD5 gene is required for error-free postreplicative bypass of UV lesions, and it encodes a DNA-dependent ATPase (6Johnson R.E. Henderson S.T. Petes T.D. Prakash S. Bankmann M. Prakash L. Mol. Cell. Biol. 1992; 12: 3807-3818Crossref PubMed Scopus (194) Google Scholar, 7Johnson R.E. Prakash S. Prakash L. J. Biol. Chem. 1994; 269: 28259-28262Abstract Full Text PDF PubMed Google Scholar). The RAD30 gene affects an alternate pathway of error-free bypass of UV lesions, and the rad5Δ rad30Δ double mutant exhibits a synergistic increase in UV sensitivity over either single mutant (8McDonald J.P. Levine A.S. Woodgate R. Genetics. 1997; 147: 1557-1568Crossref PubMed Google Scholar). Rad30 shares homology with the yeast Rev1 protein and with the Escherichia coli DinB and UmuC proteins (8McDonald J.P. Levine A.S. Woodgate R. Genetics. 1997; 147: 1557-1568Crossref PubMed Google Scholar, 9Roush A.A. Suarez M. Friedberg E.C. Radman M. Siede W. Mol. Gen. Genet. 1998; 257: 686-692Crossref PubMed Scopus (127) Google Scholar). We have recently shown thatRAD30 encodes a novel eukaryotic DNA polymerase, named Polη, which has the unique ability to efficiently replicate acis-syn thymine-thymine dimer-containing template, and it inserts two A residues across from the dimer (10Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (692) Google Scholar).The presence of biochemical activity in a protein does not necessarily imply the requirement of that activity in the biological function of the protein. For example, the deoxycytidyl transferase activity of Rev1 seems to have no role in the bypass of UV-damaged DNA templates (5Nelson J.R. Lawrence C.W. Hinkle D.C. Nature. 1996; 382: 729-731Crossref PubMed Scopus (505) Google Scholar). The S. cerevisiae Rad3 protein and its human counterpart, XPD, both possess DNA helicase activity (11Sung P. Prakash L. Matson S.W. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8951-8955Crossref PubMed Scopus (167) Google Scholar, 12Sung P. Bailly V. Weber C. Thompson L.H. Prakash L. Prakash S. Nature. 1993; 365: 852-855Crossref PubMed Scopus (283) Google Scholar), and they are required for nucleotide excision repair and for RNA polymerase II transcription. Mutational inactivation of the DNA helicase activity of these proteins, however, impairs only the repair function and not the transcription function (13Sung P. Higgins D. Prakash L. Prakash S. EMBO J. 1988; 7: 3263-3269Crossref PubMed Scopus (221) Google Scholar, 14Guzder S.N. Sung P. Prakash S. Prakash L. J. Biol. Chem. 1995; 270: 17660-17663Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 15Sung P. Guzder S.N. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 10821-10826Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Thus, the DNA helicase activities of Rad3 and XPD are required for DNA repair but not for transcription. Here, we examine whether the DNA polymerase activity of Rad30 is necessary for its role in damage bypass. For this purpose, we altered the aspartate and glutamate residues present in the highly conserved domain of serine, isoleucine, aspartate, and glutamate (SIDE) in Rad30 to alanines. The resulting Rad30 mutant protein lacks DNA polymerase activity, and this mutation inactivates the biological function of RAD30. Thus, the DNA polymerase activity of Rad30 is indispensable for its role in damage bypass. UV-induced DNA damage presents a block to the DNA replication machinery. To maintain the continuity of the DNA during replication, UV lesions encountered by the replication machinery are circumvented by both error-free and error-prone means. In the yeast Saccharomyces cerevisiae, genes in the RAD6 epistasis group function in the replication of DNA-containing lesions generated by UV light and by other DNA damaging agents. Mutations in the RAD6 andRAD18 genes confer extreme sensitivity to UV light, and these mutants are defective in postreplicative bypass of UV-damaged DNA and in UV-induced mutagenesis (1Prakash S. Sung P. Prakash L. Annu. Rev. Genet. 1993; 27: 33-70Crossref PubMed Scopus (256) Google Scholar). Rad6, a ubiquitin-conjugating enzyme, exists in vivo in a complex with Rad18, a DNA-binding protein (2Bailly V. Lamb J. Sung P. Prakash S. Prakash L. Genes Dev. 1994; 8: 811-820Crossref PubMed Scopus (277) Google Scholar, 3Bailly V. Lauder S. Prakash S. Prakash L. J. Biol. Chem. 1997; 272: 23360-23365Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). How Rad6-Rad18 protein-dependent ubiquitination promotes error-free and mutagenic postreplicative bypass is not known. Of the genes in the RAD6 epistasis group,REV1, REV3, and REV7 are required for mutagenic bypass of UV damage, and yeast lacking any of these genes is nonmutable by UV light (1Prakash S. Sung P. Prakash L. Annu. Rev. Genet. 1993; 27: 33-70Crossref PubMed Scopus (256) Google Scholar). The Rev3 and Rev7 proteins form DNA polymerase ζ, which shows limited ability to bypass acis-syn thymine-thymine dimer (4Nelson J.R. Lawrence C.W. Hinkle D.C. Science. 1996; 272: 1646-1649Crossref PubMed Scopus (595) Google Scholar). Rev1 is a deoxycytidyl transferase that can incorporate a dCMP residue opposite an abasic site (5Nelson J.R. Lawrence C.W. Hinkle D.C. Nature. 1996; 382: 729-731Crossref PubMed Scopus (505) Google Scholar). The RAD5 gene is required for error-free postreplicative bypass of UV lesions, and it encodes a DNA-dependent ATPase (6Johnson R.E. Henderson S.T. Petes T.D. Prakash S. Bankmann M. Prakash L. Mol. Cell. Biol. 1992; 12: 3807-3818Crossref PubMed Scopus (194) Google Scholar, 7Johnson R.E. Prakash S. Prakash L. J. Biol. Chem. 1994; 269: 28259-28262Abstract Full Text PDF PubMed Google Scholar). The RAD30 gene affects an alternate pathway of error-free bypass of UV lesions, and the rad5Δ rad30Δ double mutant exhibits a synergistic increase in UV sensitivity over either single mutant (8McDonald J.P. Levine A.S. Woodgate R. Genetics. 1997; 147: 1557-1568Crossref PubMed Google Scholar). Rad30 shares homology with the yeast Rev1 protein and with the Escherichia coli DinB and UmuC proteins (8McDonald J.P. Levine A.S. Woodgate R. Genetics. 1997; 147: 1557-1568Crossref PubMed Google Scholar, 9Roush A.A. Suarez M. Friedberg E.C. Radman M. Siede W. Mol. Gen. Genet. 1998; 257: 686-692Crossref PubMed Scopus (127) Google Scholar). We have recently shown thatRAD30 encodes a novel eukaryotic DNA polymerase, named Polη, which has the unique ability to efficiently replicate acis-syn thymine-thymine dimer-containing template, and it inserts two A residues across from the dimer (10Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (692) Google Scholar). The presence of biochemical activity in a protein does not necessarily imply the requirement of that activity in the biological function of the protein. For example, the deoxycytidyl transferase activity of Rev1 seems to have no role in the bypass of UV-damaged DNA templates (5Nelson J.R. Lawrence C.W. Hinkle D.C. Nature. 1996; 382: 729-731Crossref PubMed Scopus (505) Google Scholar). The S. cerevisiae Rad3 protein and its human counterpart, XPD, both possess DNA helicase activity (11Sung P. Prakash L. Matson S.W. Prakash S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8951-8955Crossref PubMed Scopus (167) Google Scholar, 12Sung P. Bailly V. Weber C. Thompson L.H. Prakash L. Prakash S. Nature. 1993; 365: 852-855Crossref PubMed Scopus (283) Google Scholar), and they are required for nucleotide excision repair and for RNA polymerase II transcription. Mutational inactivation of the DNA helicase activity of these proteins, however, impairs only the repair function and not the transcription function (13Sung P. Higgins D. Prakash L. Prakash S. EMBO J. 1988; 7: 3263-3269Crossref PubMed Scopus (221) Google Scholar, 14Guzder S.N. Sung P. Prakash S. Prakash L. J. Biol. Chem. 1995; 270: 17660-17663Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 15Sung P. Guzder S.N. Prakash L. Prakash S. J. Biol. Chem. 1996; 271: 10821-10826Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Thus, the DNA helicase activities of Rad3 and XPD are required for DNA repair but not for transcription. Here, we examine whether the DNA polymerase activity of Rad30 is necessary for its role in damage bypass. For this purpose, we altered the aspartate and glutamate residues present in the highly conserved domain of serine, isoleucine, aspartate, and glutamate (SIDE) in Rad30 to alanines. The resulting Rad30 mutant protein lacks DNA polymerase activity, and this mutation inactivates the biological function of RAD30. Thus, the DNA polymerase activity of Rad30 is indispensable for its role in damage bypass. We thank Todd Washington for discussions and Terrance Todd for the generation of therad30 Ala155-Ala156 mutation.