Yeast DNA Repair Proteins Rad6 and Rad18 Form a Heterodimer That Has Ubiquitin Conjugating, DNA Binding, and ATP Hydrolytic Activities

Abstract

The RAD6 and RAD18 genes of Saccharomyces cerevisiae are required for postreplicative bypass of ultraviolet (UV)-damaged DNA and for UV mutagenesis. The RAD6 encoded protein is a ubiquitin conjugating enzyme, and RAD18 encodes a protein containing a RING finger motif and a nucleotide binding motif. Rad18 can be co-immunoprecipitated with Rad6, indicating that the two proteins exist in a complex in vivo. Here, we co-overproduce the two proteins using a yeast multicopy plasmid, purify the Rad6-Rad18 complex to near homogeneity, and show that the complex is heterodimeric. The Rad6-Rad18 heterodimer has ubiquitin conjugating activity, binds single-stranded DNA, and possesses single-stranded DNA-dependent ATPase activity. The Rad6-Rad18 complex provides the first example wherein a ubiquitin conjugating activity is physically associated with DNA binding and ATPase activities provided by an associated protein factor. The co-existence of these activities should provide the complex with the ability to recognize single-stranded DNA resulting from stalling of the replication machinery at DNA damage sites and to recognize the components of the DNA replication machinery for ubiquitination by Rad6. The RAD6 and RAD18 genes of Saccharomyces cerevisiae are required for postreplicative bypass of ultraviolet (UV)-damaged DNA and for UV mutagenesis. The RAD6 encoded protein is a ubiquitin conjugating enzyme, and RAD18 encodes a protein containing a RING finger motif and a nucleotide binding motif. Rad18 can be co-immunoprecipitated with Rad6, indicating that the two proteins exist in a complex in vivo. Here, we co-overproduce the two proteins using a yeast multicopy plasmid, purify the Rad6-Rad18 complex to near homogeneity, and show that the complex is heterodimeric. The Rad6-Rad18 heterodimer has ubiquitin conjugating activity, binds single-stranded DNA, and possesses single-stranded DNA-dependent ATPase activity. The Rad6-Rad18 complex provides the first example wherein a ubiquitin conjugating activity is physically associated with DNA binding and ATPase activities provided by an associated protein factor. The co-existence of these activities should provide the complex with the ability to recognize single-stranded DNA resulting from stalling of the replication machinery at DNA damage sites and to recognize the components of the DNA replication machinery for ubiquitination by Rad6. Exposure of cells to ultraviolet (UV) light and to many other agents causes the formation of lesions in the DNA. During DNA replication, such lesions located in the template strand block the DNA replication machinery, resulting in a gap in the newly synthesized strand across from the damage site. A variety of postreplicational repair mechanisms have evolved to restore the continuity of the newly synthesized DNA strand (reviewed in Ref. 1Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society for Microbiology Press, Washington, D. C.1995Google Scholar). Genetic studies in the yeast Saccharomyces cerevisiae have been instrumental in identifying the genes involved in postreplicational repair. RAD6 and RAD18, members of the RAD6 epistasis group, play a prominent role in this repair process. Mutations in RAD6 cause extreme sensitivity to UV light and to other DNA damaging agents; rad6 mutants are highly deficient in postreplicational repair of UV-damaged DNA (2Prakash L. Mol. Gen. Genet. 1981; 184: 471-478Crossref PubMed Scopus (234) Google Scholar) and they exhibit no mutation induction in response to UV (3Lawrence C.W. Adv. Genet. 1982; 21: 173-254Crossref PubMed Scopus (139) Google Scholar).RAD6 encodes an ubiquitin conjugating enzyme of 172 residues (4Jentsch S. McGrath J.P. Varshavsky A. Nature. 1987; 329: 131-134Crossref PubMed Scopus (546) Google Scholar, 5Sung P. Prakash S. Prakash L. Genes Dev. 1988; 2: 1476-1485Crossref PubMed Scopus (142) Google Scholar). The first 149 amino acids of Rad6 form a globular domain, while the distal 23 residues, which are predominantly acidic, constitute a freely extending tail domain (6Morrison A. Miller E.J. Prakash L. Mol. Cell. Biol. 1988; 8: 1179-1185Crossref PubMed Scopus (78) Google Scholar). Mutational inactivation of the active site cysteine 88 residue in Rad6 has indicated that the ubiquitin conjugating activity is essential for all the biological functions of Rad6 (7Sung P. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2695-2699Crossref PubMed Scopus (128) Google Scholar). Mutants of RAD18 resemble those of RAD6in their high degree of sensitivity to UV, defects in postreplicational repair of UV-damaged DNA (2Prakash L. Mol. Gen. Genet. 1981; 184: 471-478Crossref PubMed Scopus (234) Google Scholar), and defects in UV mutagenesis (8Cassier-Chauvat C. Fabre F. Mutat. Res. 1991; 254: 247-253Crossref PubMed Scopus (57) Google Scholar, 9Armstrong J.D. Chadee D.N. Kunz B.A. Mutat. Res. 1994; 315: 281-293Crossref PubMed Scopus (27) Google Scholar). However, unlike RAD6, which is indispensable for sporulation, mutations in RAD18 do not affect sporulation (10Jones J.S. Weber S. Prakash L. Nucleic Acids Res. 1988; 16: 7119-7131Crossref PubMed Scopus (112) Google Scholar). Other genes that belong to the RAD6 epistasis group includeREV1, REV3, REV7, and RAD5. Although mutants of the REV genes show only a marginal increase in UV sensitivity, like rad6 and rad18 mutants, they are defective in UV mutagenesis (3Lawrence C.W. Adv. Genet. 1982; 21: 173-254Crossref PubMed Scopus (139) Google Scholar, 11Lemontt J.F. Genetics. 1970; 68: 21-33Crossref Google Scholar, 12Lawrence C.W. Nisson P.E. Christensen R.B. Mol. Gen. Genet. 1985; 200: 86-91Crossref PubMed Scopus (47) Google Scholar). Rev3 and Rev7 together form a DNA polymerase activity (polζ) that can bypass a thymine-thymine cis-syn-cyclobutane dimer (13Nelson J.R. Lawrence C.W. Hinkle D.C. Science. 1996; 272: 1646-1649Crossref PubMed Scopus (598) Google Scholar). Mutations inRAD5 enhance UV sensitivity to a greater degree than those in the REV genes; however, the incidence of UV mutagenesis at most loci is not affected (14Johnson R.E. Henderson S.T. Petes T.D. Prakash S. Bankmann M. Prakash L. Mol. Cell. Biol. 1992; 12: 3807-3818Crossref PubMed Scopus (195) Google Scholar). From these and other genetic observations, it has been suggested that REV genes andRAD5 function, respectively, in the mutagenic and nonmutagenic modes of RAD6, RAD18-dependent postreplicational repair. Rad18 can be co-immunoprecipitated with Rad6, indicating physical interaction of the two proteins (15Bailly V. Lamb J. Sung P. Prakash S. Prakash L. Genes Dev. 1994; 8: 811-820Crossref PubMed Scopus (279) Google Scholar). For delineating the molecular functions of the Rad6-Rad18 complex in postreplicative repair processes, it is essential to purify this complex and to define its biochemical properties. Here, the Rad6-Rad18 complex is purified to near homogeneity from yeast cells genetically tailored to co-overproduce the two proteins. We show that the Rad6-Rad18 complex is heterodimeric and that the Rad6-Rad18 complex has ubiquitin conjugating activity, as well as single-stranded (ss) 1The abbreviations used are: ss, single-stranded; ds, double-stranded; DTT, dithiothreitol; GST, glutathioneS-transferase; ATPγS, adenosine 5′-O-(thiotriphosphate). DNA binding and ssDNA-dependent ATPase activities. The DNA fragment with the ADC1 promoter and the RAD6 gene from pSCW242 (5Sung P. Prakash S. Prakash L. Genes Dev. 1988; 2: 1476-1485Crossref PubMed Scopus (142) Google Scholar) was cloned in a derivative of a 2-μm multicopy vector, which contains the GAL1 promoter and the URA3 gene. A DNA fragment containing theRAD18 gene from the ATG initiation codon to nucleotide 342 after the termination codon was cloned under the GAL1promoter in the vector containing the ADC1-RAD6 insert. The plasmid obtained, pR18.36, is shown in Fig. 1 A. Yeast strain LY2 (MATα leu2–3, leu2–112 gal1 reg1–501 pep4–3 ura3–52 trp1Δ prb112), harboring the Rad6-Rad18 overexpressing plasmid pR18.36 (2 μm, ADC1::RAD6 GAL1::RAD18), was grown at 30 °C to midlogarithmic phase (1 × 107 cells/ml) in synthetic complete medium lacking uracil and diluted with 10 volumes of YPD (yeast extract-peptone-dextrose) supplemented with 1% galactose. The culture was incubated in 10-liter batches in fermentors until a cell density of ∼2 × 108 cells/ml was reached (∼12 h). Cells were harvested by centrifugation, washed with ice-cold distilled water, frozen in liquid nitrogen, and stored at −70 °C. All subsequent steps were carried out at 4 °C. To prepare cell extract, frozen yeast cells (60 gm) were thawed by stirring in 50 mm Tris/HCl, pH 7.5, 100 mmNaCl, 1 mm EDTA, 10 mm β-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin, leupeptin, chymostatin, and aprotinin (3 ml of buffer/g of cells) and lysed by passage through a French press at 16,000 p.s.i. The supernatant obtained by centrifugation at 20,000 × gfor 30 min was brought to 30% saturation with ammonium sulfate. A second ammonium sulfate precipitation was carried out by bringing the supernatant recovered by high speed centrifugation (100,000 ×g for 1 h) to 50% saturation with ammonium sulfate. The pellet was kept on ice overnight and then dissolved in 50 mm Tris/HCl, pH 7.5, 1 mm EDTA, 5 mm DTT, NaCl buffer containing protease inhibitors to give an ionic strength equivalent to that of 0.2 m NaCl in water. The protein solution was applied onto a Q-Sepharose FF (Pharmacia Biotech Inc.) column (60 ml) equilibrated in buffer A containing 0.2 m NaCl (buffer A: 50 mmTris/HCl, 1 mm EDTA, 2 mm DTT, pH 7.5) and washed with 180 ml of buffer A containing 0.25 m NaCl. The Rad6-Rad18 complex was eluted with buffer A containing 0.4m NaCl. The sample (30 ml) was applied to a phenyl-Sepharose CL-4B (Pharmacia) column (20 ml) equilibrated with buffer A containing 0.4 m NaCl and washed after sample application with the same buffer. The flow-through and first 20 ml of the wash were pooled (50 ml), and proteins were precipitated with ammonium sulfate to 50% saturation. The pellet was dissolved in buffer A containing 0.15 m NaCl. Insoluble material was removed by centrifugation, and the sample was applied onto a Mono S column (HR5/5; Pharmacia) equilibrated with buffer B containing 0.15 mNaCl (buffer B: 50 mm Tris-HCl, 0.1 mm EDTA, 1 mm DTT, pH 7.5). The column was developed with a 20-ml NaCl gradient from 0.2 m to 0.4 m, and fractions containing the Rad6-Rad18 complex, which elutes at about 0.3m NaCl, were pooled, diluted twice with buffer C (buffer B containing 10% glycerol), and applied onto a Mono Q column equilibrated with buffer C containing 0.15 m NaCl. Rad6-Rad18 complex was eluted with a 20-ml NaCl gradient from 0.2m to 0.5 m, and the peak fractions, at about ∼0.38 m NaCl, were pooled. The concentration of Rad6-Rad18 in the final sample was determined from the absorbance at 280 nm using an extinction coefficient of 56800m−1 cm−1, calculated according to the formula (number of tryptophan residues × 5700) + (number of tyrosine residues × 1300). Rad6-Rad18 complex obtained from this purification scheme (∼5 mg) was stored in small portions at −70 °C. A 200-μl portion of the Mono Q pool (450 μg of Rad6-Rad18) was concentrated to 20 μl with a microcon 30 (Amicon), diluted to 100 μl with 25 mmHEPES/KOH, pH 7.0, 50 mm KOAc, 0.5 mm EDTA, 1 mm DTT, and loaded onto a 11.7-ml glycerol gradient (3.5–27.5%) made in the same buffer. Molecular weight standards (catalase, aldolase, bovine serum albumin, ovalbumin, chymotrypsin) were loaded on a parallel, identical gradient. After centrifugation (24 h at 40,000 rpm and 4 °C in a SW 41 rotor), 0.5-ml fractions were collected and analyzed by SDS-polyacrylamide gel electrophoresis. The EcoRI fragment from YCpUBA1 (16McGrath J.P. Jentsch S. Varshavsky A. EMBO J. 1991; 10: 227-236Crossref PubMed Scopus (189) Google Scholar), which contains the UBA1 gene in theCEN URA3 plasmid YCp50, was cloned into a yeast 2-μm multicopy vector, yielding plasmid pPM196. Three chromatographic steps, consisting of Q-Sepharose, ubiquitin-Sepharose, and Mono Q, adapted from (5Sung P. Prakash S. Prakash L. Genes Dev. 1988; 2: 1476-1485Crossref PubMed Scopus (142) Google Scholar), were used for purification of Uba1 to apparent homogeneity from yeast strain LY2 carrying plasmid pPM196. Rad6 was purified from yeast strain CMY135 carrying plasmid pSCW242 (ADC1::RAD6), as described previously (7Sung P. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2695-2699Crossref PubMed Scopus (128) Google Scholar). GST-ubiquitin (17Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1978) Google Scholar) was prepared and as described W. F. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). (1 was incubated with Uba1 Rad6 Rad6-Rad18 in 10 μl of 25 mm pH 7.5, 25 mm 5 mm 2 mm 0.1 mm The was by the of 10 μl of buffer mm Tris/HCl, pH m of sample was with 1 μl of 1 m and for the were incubated for min at 30 °C. were to SDS-polyacrylamide gel and analyzed by ubiquitin was prepared as described A. S. A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). 0.5 was incubated for min at 30 °C with Uba1 Rad6 Rad6-Rad18 in 50 mm Tris/HCl, pH 7.5, 50 mm 5 mm 2 mm 0.2 mm DTT, 100 μg/ml bovine serum The were on a The gel was and to DNA binding was using the binding were in 0.4 m for washed in distilled water, and stored in buffer mm pH 7.0, mm prepared in this be used a of at 1 The buffer for DNA binding of + 50 μg bovine serum and 0.5 mm (Pharmacia) was dissolved in mm Tris/HCl, pH 7.5, 0.5 mm and used as a concentrated the of the was The DNA was with by first with and then with and DNA was used at a concentration of 2 was to 4 mm and was to 1 mm containing the DNA and the Rad6-Rad18 complex were incubated for 10 min at 30 and two from sample were on which were washed with 2 ml of and the associated by the two for by All were at obtained on the same are by the Rad6-Rad18 complex was using on The buffer used for the of 25 mm pH 7.0, 50 4 mm 50 μg/ml bovine serum albumin, and 0.5 mm was to a concentration of 5 of were to DNA and protein are indicated in the were by the of (2 were removed at and the by the of an of 20 mm EDTA, A portion of sample (1 was onto a that been in 0.5 1 m and in distilled and The was developed in the and the of in a were to and the resulting calculated by The the of at two To of Rad6 and Rad18 proteins in we plasmid pR18.36 in which the RAD6 gene is to promoter and the RAD18 gene is to promoter 1 pR18.36 in and of Rad6 and Rad18 Rad6 and Rad18 exist in a complex and were as such to near homogeneity by a of ammonium sulfate precipitation and column chromatographic steps of Q-Sepharose, Mono and Mono Q and and Fig. 1 The Rad6 and Rad18 proteins associated through all these purification steps C and indicating a high degree of of the of a sample of purified Rad6-Rad18 complex suggested that the proteins were in To the of the complex, a sample of the Mono Q was to phase on a column to Rad6 and Rad18 by of the absorbance of protein peak at and 280 The of the absorbance of Rad18 to the absorbance of Rad6 at were with the for a complex The were to the for a complex, indicating that Rad6 and Rad18 are in the To the of the Rad6-Rad18 complex, a of the Mono Q sample was analyzed by in a glycerol we that Rad6-Rad18 with an apparent molecular of the that Rad6 and Rad18 to form a highly Rad6-Rad18 complex has a of protein complex was on a phase the of the two proteins was by UV absorbance at and 280 nm and by an by gel the under the to Rad6 and Rad18 and the were calculated the of amino acids in protein was used to the at The 280 of × 5700) + of × was used to the at 280 in a Rad6-Rad18 protein complex was on a phase the of the two proteins was by UV absorbance at and 280 nm and by an by gel the under the to Rad6 and Rad18 and the were calculated the of amino acids in protein was used to the at The 280 of × 5700) + of × was used to the at 280 Although Rad6 protein can be purified (5Sung P. Prakash S. Prakash L. Genes Dev. 1988; 2: 1476-1485Crossref PubMed Scopus (142) Google Scholar), we have not in Rad18 protein by no of Rad18 be obtained yeast containing Rad18 protein was to in chromatographic Q-Sepharose, and single-stranded DNA not The chromatographic of Rad18 protein that of the protein be its with Rad6 Rad6 protein has ubiquitin conjugating activity (4Jentsch S. McGrath J.P. Varshavsky A. Nature. 1987; 329: 131-134Crossref PubMed Scopus (546) Google P. Prakash S. Prakash L. Genes Dev. 1988; 2: 1476-1485Crossref PubMed Scopus (142) Google Scholar). We the Rad6 ubiquitin conjugating activity was affected of the with Rad18 by the ability of Rad6 protein to form a with ubiquitin 2 and to the ubiquitin to a protein 2 a can be by with a such as β-mercaptoethanol, to formation of a ubiquitin and Rad6, the of two and with in the of were 2 to this GST-ubiquitin was used it be W. F. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). the of and the enzyme Rad6 a with 2 Fig. 2 Rad6 a with in of Uba1 and and the to the same with Rad6 2 and the binding of Rad18 to Rad6 not the from with Uba1 and a with (5Sung P. Prakash S. Prakash L. Genes Dev. 1988; 2: 1476-1485Crossref PubMed Scopus (142) Google Scholar) was used as the to the ability of Rad6 to the formation of ubiquitin with protein The in Fig. 2 that is a for Rad6, indicating that the Rad6-Rad18 complex ubiquitin conjugating activity. We used binding to the interaction the Rad6-Rad18 heterodimer and DNA. this protein DNA are on the DNA through the of the of DNA by Rad6-Rad18 complex by the associated with the The in Fig. show that the Rad6-Rad18 complex binds the single-stranded in a protein of the the at 0.25 Rad6-Rad18 We determined the of the Rad6-Rad18 complex for and DNA. was carried out by the ability of DNA to with for binding to the Rad6-Rad18 The in Fig. B were obtained from wherein 0.1 Rad6-Rad18 complex was first incubated with for 10 and of were as After a the were applied to and the of with Rad6-Rad18 complex the of 5 binding to the by as as DNA not binding of the that Rad6-Rad18 complex binds binds only binding studies and that Rad6-Rad18 has for not the is carried out in the with the Rad6-Rad18 complex to the of the binding to The of is than that for not with the for by the Rad6-Rad18 we that the Rad6-Rad18 complex has a for binding to and to in as the same is DNA is used in of not the Rad6-Rad18 complex a ssDNA-dependent ATPase activity it was of to the DNA binding of the Rad6-Rad18 However, have no on the binding of not We the of in two other which were to in DNA binding as DNA we first for in the sensitivity to by of NaCl, and at the of from a The of has no on the sensitivity to 4 on the of from 4 we that the DNA binding of the Rad6-Rad18 complex is not by The Rad18 protein contains a Walker A nucleotide binding motif in a variety of proteins that and (10Jones J.S. Weber S. Prakash L. Nucleic Acids Res. 1988; 16: 7119-7131Crossref PubMed Scopus (112) Google Scholar). it was to the Rad6-Rad18 complex has ATPase activity. We that purified Rad6-Rad18 complex with a on a DNA with single-stranded DNA than DNA in ATPase activity. For Rad6-Rad18 ATPase activity was by only by DNA. the fractions from the chromatographic purification in Mono Q were for their ATPase activity, of the ATPase activity with the Rad6-Rad18 complex was We a sample of the Mono Rad6-Rad18 complex to molecular in a column and the of this ATPase activity with the Rad6-Rad18 protein not The and the high degree of of the Rad6-Rad18 complex that the ATPase activity is an of this protein The concentration of the activity in this the of is in the Rad6-Rad18 complex, the is to the of The of are a 1 h the concentration of of the nucleotide is no by is no of that the Rad6-Rad18 complex the of to and We carried out a protein in the of a of while the The in Fig. B show that the of with protein until an is reached all of the binding sites are that all of the sites on the DNA are the site calculated for the Rad6-Rad18 complex the binding site is calculated from these by the protein concentration at the is in with the calculated the of DNA is well in of was to all the protein to The pH of the activity that pH and pH is no in the of not this we show that Rad18 in a complex with Rad6. The two proteins through chromatographic in of Q-Sepharose, Mono and Mono by phase and glycerol gradient the purified complex contains an of Rad6 and The Rad6-Rad18 complex the first example wherein a ubiquitin conjugating enzyme is physically associated with a provides an for the and RAD18 in DNA repair and mutagenesis. The Rad6-Rad18 complex a with ubiquitin and ubiquitin to the complex the ubiquitin conjugating activity of Rad6. we that the Rad6-Rad18 complex binds to Rad6 contains no DNA binding and no DNA binding the binding ability of the Rad6-Rad18 complex from with Rad18 contains a motif (10Jones J.S. Weber S. Prakash L. Nucleic Acids Res. 1988; 16: 7119-7131Crossref PubMed Scopus (112) Google Scholar), as the RING finger motif Biol. Sci. 1996; 21: Full Text PDF PubMed Scopus Google Scholar), as well as a motif (10Jones J.S. Weber S. Prakash L. Nucleic Acids Res. 1988; 16: 7119-7131Crossref PubMed Scopus (112) Google Scholar), of which be in DNA The Rad6-Rad18 complex an ssDNA-dependent ATPase activity with a of The high degree of of the complex and the of the ATPase activity with the Rad6-Rad18 complex that this activity is to this The of the Walker A nucleotide binding motif in Rad18 (10Jones J.S. Weber S. Prakash L. Nucleic Acids Res. 1988; 16: 7119-7131Crossref PubMed Scopus (112) Google Scholar), and the of nucleotide binding motif of activity in Rad6 that the ATPase activity of the complex in The that the ATPase activity is that the activity is a and only in the of an as is the in a to that with the of ATPase activity by cell Proc. Natl. Acad. Sci. U. S. A. 1990; 87: PubMed Scopus Google Scholar). the DNA and ubiquitin conjugating activities of the Rad6-Rad18 complex in the postreplicative bypass of The DNA binding activity the complex to sites of the DNA replication machinery has been by DNA the ATPase activity to have no on the DNA binding activity of the Rad6-Rad18 complex, it is that this activity is in role as a molecular A. Science. 1993; PubMed Scopus Google Scholar), wherein Rad18 the protein for ubiquitination by Rad6, and binding and these The of the purified Rad6-Rad18 complex, and the of biochemical activities the to the components of the replication machinery that are by the complex and to of of the proteins is required for the of the postreplicative bypass DNA repair We P. Sung for