The RAD6 gene of Saccharomyces cerevisiae functions in postreplication repair of ultraviolet damaged DNA, and interestingly, it also has a role in increasing ...
Vol. 269, No. 45, Issue of November 11, pp. 28259-28262, 1994 Printed in U.S.A.
CHEMISTRY THE JOURNALOF BIOLOGICAL 0 1994 by The American Society for’ Biochemistry and Molecular Biology, Inc.
Yeast DNA Repair ProteinR A D 5 That Promotes Instabilityof Simple Repetitive Sequencesis a DNA-dependent ATPase* (Received for publication, July 26, 1994, and in revised form, August 23, 1994)
Robert E. JohnsonSQ,Satya PrakashQ,and Louise PrakashSQn From the Department of Biophysics, University of Rochester, Rochester, New York a n d the SSealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Medical Research Building, Galveston, Texas 77555-1061
The RAD6 gene of Saccharomyces cerevisiaefunctions functions predominantly in error free postreplication repair in postreplication repair of ultraviolet damaged DNA, a n d R E V land REV3 carry outthe mutagenic bypassof lesions and interestingly, it also has a role in increasing the in the template strand (5). in the genome. instability of simple repetitive sequences Interestingly, in addition toits role in postreplication repair, In contrast to DNA mismatch repair genes which func- RAD5 affects the stability of simple repeat sequences (5). In tion in maintaining constant length of repeat sequences, humans, expansion of trinucleotide repeats is associated with RAD5 promotes alterations in the length of repeat se- at least seven neurological disorders including fragile X synquences. In this work, we purify the R A D 5 protein to drome,Huntington’sdisease, and myotonicdystrophy (6). near homogeneity from yeastcells and show thatit is a Widespread alterationsin lengths of simple repeated sequences single-stranded DNA-dependent ATPase. The possible also occur in tumors from hereditary nonpolyposis colorectal roles of RAD6 ATPase in postreplication gap filling andcancers (7-9) and in pancreatic and gastric carcinomas (10,ll). inincreasingtheincidence of lengthalterations of In yeast, mutations in DNA mismatch repair genes cause inrepeat sequences are discussed. creased instability of repeated sequences (12), whereas mutations in the R A D 5 gene render these sequences more stable (5). Thus, in wild type cells, mismatch repair genes help maintain Replication of a DNA template containing cyclobutane py- constant length of repetitive tracts, while R A D 5 promotes tract rimidine dimers and other typesof damage caused by ultravio- length alterations. let irradiation results in the formation of a g a p in the newly Here, we purifythe RAD5 protein from yeastand show that synthesized DNA strand because of the inability of DNA po- it has a single-stranded DNA-dependentATPase activity. Even sites. The gap is then though RAD5 contains all of the conserved domains foundaslymerases to replicate past such damage filled in by postreplication repair mechanisms. Genetic studies sociated with DNA helicases, we find no evidence of a DNA in yeast have suggested the involvement of genes belonging to helicase activity in this protein. the R A D 6 epistasis group, W 6 , RAD18, RAD5, REVl, and EXPERIMENTALPROCEDURES REV3, in the postreplication repair pathway (1). Mutations in Anti-RAD5 Antibodies-Anti-RAD5 antibodies were generated in RAD6 and RAD18 render cells highly sensitive to ultraviolet against aportion of the R A D 5 protein encompassing amino acid light, and they confer a defect in postreplication repair of UV- rabbits residues 233to 801(5) fusedto the first 48 amino acids of the damaged DNA (2) and in UV mutagenesis (1,3).RAD6 encodes Escherichia coli transcription termination protein p. Polyclonal antia ubiquitin-conjugating enzyme a n d it forms a tight complex bodies wereaffinity purified from serum using a Sepharose column with with the RAD18 protein (4). Genetic studies have implicated covalently coupled pRAD5 fusion protein. Expression and Purification of RAD5 from Yeast-To overexpress the requirement of the RADG.RAD18 complex in DNA repair. We have suggested that complex formation providesa mecha- RAD5 in yeast, the RAD5 gene from position -37 to +3966 (5) was nism by whichRAD6 ubiquitin-conjugating activity is targeted placed downstream of the ADCl promoter in plasmid pSCW231, generating pBJ29. Yeast strain LP2749-9Bharboring pBJ29 was grownin t o the DNA damage sites via the DNA binding activity of 12-liter batches in synthetic medium lacking tryptophan. Extract was RAD18 (4). prepared from 350 g of frozen yeast cells by using a French press and R A D 5 and REV3 constitute alternate routes of DNA repair clarified by centrifugation at 110,000 x g for 1.5 h. Solid ammonium controlled by the RADG/RAD18 proteins ( 5 ) .A synergistic in- sulfate was added to 35% saturation (0.21 g/ml of extract) and precipicrease in UV sensitivity occurs when the rad5A mutation is tated protein was pelleted at 16,000 x g. The protein pellet was resuscombined with either the revlA or the rev3A mutation. Also, pended in 600 mlof buffer A (20 mM KH,PO,, pH 7.4, 10%glycerol, 0.5 mM EDTA, 10 mM 2-mercaptoethanol, 0.5 mM phenylmethylsulfonyl whereas the REVl and REV3 genes are required for UV mu- fluoride, and 0.5 mM benzamidine hydrochloride) supplemented with 10 tagenesis, RAD5has only a marginal effect onUV mutagenesis pg/ml aprotinin, chymostatin, leupeptin, and pepstatin A and dialyzed (5):UV-induced forward mutationsat the CANl‘ locus occurat against 10 volumes of buffer A until the conductivity of the protein the normal ratein the rad5A mutant, and UV induced rever- solution was the same as thatof buffer A with 50 m KC1. The protein sion of certain ochre mutations is reduced onlya few fold by the suspension was then loaded ontoa 320-ml S-Sepharosecolumn bygravrad5A mutation. These studieshavesuggested that RAD5 ity, whichwas washed with 800 ml ofdialysis buffer and eluted with 300 n m KC1 in buffer A. Fractions containing protein were identified by the Coomassie Blue dye binding assay,pooled, and dialyzed against 10 * This work was supported by National Institutes of Health Grant volumes buffer A for 9 h. The dialysate was loaded onto a 50-ml SGM19261. The costs of publication of this article were defrayed in part Sepharose column and washed with 3 column volumesof buffer A conby the payment of page charges. This article must therefore be hereby taining 25 m KC1 before developingwith a 900-ml gradient of 25-275 mMKC1 in buffer A. RAD5-containingfractions were identified by immarked “advertisement” in accordance with 18 U.S.C.Section1734 munoblotting and pooled before dialysis against 10 volumes of buffer A solely to indicate this fact. llTo whom correspondence should be addressed: Sealy Center of for 1 h. The pool was then loaded onto an 8-ml Q-Sepharose column, Molecular Science,University of Texas Medical Branch, 6.104 Medical washed with 5 column volumesof 50 mM KC1 in buffer A, and developed Research Bldg., 11th and Mechanic St., Galveston,TX 77555-1061. Tel.: with a 120-ml50-400 m KC1 gradient in buffer A. Fractions containing 409-747-8602;Fax: 409-747-8608. RAD5 were identified by immunoblotting,pooled, and dialyzed against
28259
R A D S is a DNA-dependent ATPase
28260
A
M,(K)
B
1 2 3
200 -
M,(K)
200-
116-
C
Fraction
97 -
116-
66 -
66-
45 -
45-
97
-
15
-
10
-
-
-
D
9 10 1 1 12 13 1 4 1 5 1617 18
*RADS
lmmunoblot
20
12 3
0 5 -
0.5
1.0 -
13
2.0
pg antibody
09
10
11 12
13 14
15
16
17 18
Fraction FIG.1.Purification of R A D 5 protein and identification of its ssDNA-dependent ATPaseactivity.A, overexpression and purification of RAD5 protein. Nitrocellulose blotof a n 8% denaturing polyacrylamidegel was probed with antibodiesspecific for RAD5. Lane 1,extract from yeast strain LP2749-9B containing the vector pSCW231 without the RAD5 insert. Lane 2, extract from strain LP2749-9B containing the RAD5 overexpressing plasmid pBJ29. Lane 3, 10 ng of purified RAD5 protein. B, Coomassie Blue-stained 8% denaturing polyacrylamidegel. Lane 1, molecular weight standards. Lane 2, 250 ng of RAD5 protein. Lane 3, 1 pg of RAD5 protein. C,ATPase activity coelutes with RAD5 protein. Fractions 9-18, 1 pl each, from the final hydroxylapatite chromatography column were assayed for ATPase activity, and subjected to immunoblotting to examine their RAD5 content. Closed circles, ATPase activity in fractions obtained from LP2749-9B containing theRAD5 overexpressing plasmid pBJ29; open circles, ATPase activity in fractions obtained from yeast strain LP2749-9B containing the vector with no RAD5 insert. D , specific inhibition of RAD5 ATPase activity by anti-RAD5 antibodies. Increasing amounts of either anti-RAD5(open circles) or anti-REV3(closed circles) antibodies were added to the standard ATPase reaction mixture. a Bio-Gel HTP 100 volumes buffer for A 1h prior to loading onto1.5-ml hydroxylapatite (Bio-Rad) column. The column was washed with5 column volumes of buffer B (buffer A containing 50 mM KC1 and 1 mM dithiothreitol) and eluted with a0-250 mM KH,PO, gradient in buffer B. RAD5 containing fractions were concentrated using Centricon 30 microconcentrators (Amicon) and stored in 15-pl aliquotsa t -70 "C. ATPase Assays-Unless otherwise stated, purifiedRAD5 protein (50 ng) was incubated in 10-pl reactions containing mM Tris-HC1, 20 pH 7.0, 20 mM KCl, 2 mM MgCl,, 100 pg/ml bovine serumalbumin, 1 mM dithiothreitol, 0.5 mM [2,5,8-3H]ATP,and 200 ng of DNA for 25 min a t 30 "C. ATPase activity was measuredby thin layer chromatography on polyethyleneimine-celluloseand scintillation counting. DNA Helicase Assays-DNA helicase assays(10pl) were carried out under conditions similar to ATPase assays except that ATP was increased to 2mM, and 2-5 ng of 32P-labeled DNA substrate replaced 200 ng of ssM13' DNA. Reactions were carried out for 30 min a t 30 "C. Reaction products were analyzed by electrophoresis in nondenaturing polyacrylamide gelsfollowed by autoradiography. Theeffect of pH was assayed by using the following buffers: pH 5.0-6.5, KMES; pH 7.0-8.5, Tris-HCl; pH 9.0-10.0, CAPS. The abbreviationsused are: ss, single-stranded; CAPS, 3-(cyclohexylamino)propanesulfonic acid; KMES, potassium 2-[N-morpholinolethanesulfonic acid.
RESULTS
Purification of RAD5 Protein-For purification of the RAD5 protein from yeast, we placed the RAD5 gene under thecontrol of the constitutivealcohol dehydrogenase (ADCl ) promoter in the 2-pm multicopy vector pSCW231 to yield plasmid pBJ29. Immunoblotting with affinity purified antibodies specific for RAD5 protein reveals a much higher level of RAD5 protein in yeast strain LP2749-9B harboring pBJ29 than in LP2749-9B harboring thevector pSCW231 (Fig. lA,compare lane 2 to lane 1 ). RAD5 is of low abundance inwild type cells (Fig. L4, lane 11, and itsdetection requires enrichmentby immunoprecipitation. RAD5 protein in the overproducing extract (Fig. LA, lane 2 and R A D 5 immunoprecipitated from wild type extract (data not shown) both show a n molecular mass of 139 kDa when analyzed by SDS-polyacrylamide gel electrophoresis, which is consistent with the size of 134 kDa predicted by DNA sequence analysis (5).RAD5 was purified to nearhomogeneity (Fig. LB), and the identity of pure protein verified by immunoblotting (Fig. L4, lane 3).
RAD5 is a DNA-dependent ATPase
28261
TABLEI RAD5 has ssDNA-dependent ATPase activity ATPases assays were carried out as described under “Experimental Procedures.”2 m~ Ca2+,Mn2+,or Zn2+ were used to replace M e . 200 ng of the indicated polynucleotide was used and reactions were carried out for 25 min at 30 “C. Conditions
ATP hydrolyzed %
No Single-stranded DNA M13
”e - M e + Ca2+
- M e + Mn2+ - M e + Zn2+ +X174 Double-stranded DNA closed pBR322, circular pBR322, linear RNA Poly(A) Poly(U) Total yeast RNA
0
38 0 37 32 0 45 3.1 3.3
PH
8
100-
0.9 0.8 5.5
ATPase Activityof RADS-Purified RAD5 protein hydrolyzes ATP to ADP and Pi in thepresence of single-stranded (ss) DNA (Table I). Little ATPase activity is observed with doublestranded DNA (Table I), and UV irradiation ofDNA has no effect on the ATPase activity (data not shown). RNA is a poor cofactor for ATP hydrolysis by RAD5 (Table I). RAD5 ATPase activity requires M e , which can be substituted by Mn2+and I). RAD5 hydrolyzes dATP with the Ca2+,but not by Zn2+ (Table same efficiency and cofactor requirements as ATP. Other NTPs and dNTPs were not significantly hydrolyzed by RAD5 (data not shown). The very high degree of purity of RAD5 protein (Fig. LB) strongly suggested that theATPase activity is intrinsic to this protein. To confirm this, we determined whether ATPase activity copurifies with the RAD5 protein and whether the activity can be specifically inhibited by anti-RAD5 antibody.As shown in Fig. lC, thessDNA-dependentATPase activity coeluted with RAD5 protein during the final hydroxylapatite chromatography step. This activity was absent in equivalent fractions derived from theextract of yeaststrain carrying the vector pSCW231 lacking RAD5 (Fig. lC), and no RAD5 could be detected in these fractions by immunoblotting (data not shown). Fig. lD shows that ATPase activity is strongly inhibited by anti-RAD5 antibodies but not by antibodies raised against the unrelated yeast REV3 protein. These results establish that the ssDNA-dependentATPase activity is intrinsic to RAD5. RAD5 ATPase has a pH optimum of 7.0 (Fig. 2 A ) . As shown in Fig. 2, B and C, RAD5 catalyzes ATP hydrolysis in a proteinand time-dependent fashion. RAD5 has aK,,, of 525 p~ ATP at pH7.0 as determined by Lineweaver-Burk analysis using ssM13 DNA. RAD5 Shows No DNA Helicase Activity-Many ssDNA-dependent ATPases have an associated DNA helicase activity. The presence of conserved helicase domains in RAD5 (5) suggested that itmay also contain DNA helicase activity. We used several DNA substrates to assay for DNA helicase activity in RAD5 These included 17-mer and 41-mer oligonucleotides annealed to M13 circular ssDNA, a linear partial duplex in which a 17-mer oligonucleotide was hybridized in the middle of a 200nucleotide long ssDNA,and atailed substrate that contained a 69-base pair duplex region and single-stranded tails of 188 and 377 nucleotides, respectively, obtained by annealing ssDNA to circular ssM13 DNA.However,we found no evidence of unwinding of any of these substrates by RAD5 under a variety of
I
0
100
200
300
RADS (ng)
0
0
20
40
60
Time (min) FIG.2. Characterization of R A D S ATPase activity. A, pH dependence of RAD5 ATPase activity. Buffers were as follows; pH 5.0-6.5, KMES; pH 7.0-8.5 Tris-HC1; pH9.0-10.0, CAPS. B and C, ATP hydrolysis as a function of RAD5 protein concentration and reaction time.
assay conditions, including the addition of yeast singlestranded DNA-binding protein RPA. DISCUSSION
RAD5 ATPase couldfunction in postreplication repair and in tract-length alterations by increasing the processivity of DNA polymerase or by effecting the dissociation of the primer strand from the template. Error free replicative bypass of T-T cis-syn dimer occurs with a high frequency in Saccharomyces cerevisiae (13). RAD5 ATPase could be instrumental in this process by increasing the processivity of DNA polymerase. No mutations would occur if insertion of A residues occurs opposite the T-T dimer, as expected from the “ A rule (14). The T subunit of E. coli DNApolymerase I11 holoenzyme is asingle-stranded DNAdependent ATPase and it increases the processivity of polymerase I11 (15, 16). In bacteriohage T4, the accessory proteins
28262
RAD5 is a DNA-dependent ATPase
gp44, gp62, and gp45 together comprise an ATPase activity which increases the rate and processivity of DNA polymerase (17, 18). A processivity role of RAD5 could also explain the involvement of this protein in increasing the rate of length alterations of simple repeat sequences, where RAD5 could enhance the replication of a DNA template containing a loop out of nucleotides in the template or the primer strand resulting from DNA strand slippage. RAD5 would then affect the tract length instability by increasing the usage of misaligned DNA as a substrate for continued replication. Alternatively,RAD5 may function in thebypass of DNAdamage by a copy choicemechanism of DNA synthesis in which the 3' end of the growing DNA strand separatesfrom the damaged template and utilizes the undamaged strand in the sister duplex as the template for DNA synthesis (19, 20). Following replication past the DNA damage, the newly synthesized DNA strand reanneals back to the damaged DNA template. Even though RAD5 alone shows no DNA unwinding activity, in conjunction with other protein(s), RAD5 may unwind the growing DNA strand from the damaged DNA template and promote branch migration in copy-choice type ofDNA synthesis. The dissociation of the primer strand from the template strand could also account for the role of RAD5 in decreasing the stability of simple repetitive sequences. In situations where DNA replication is blocked due to the presence of sites in the template strand that terminate processive synthesis, RAD5 ATPase in concert with other proteins could destabilize the annealed 3' end from the template strand. In regions of the genome containing repetitive sequences, the primer could then reassociate with the template strand in a misaligned fashion and be used for DNA synthesis. Elongation of the tract would result if a loop out of nucleotides occurs in the primer strand and shortening of the tract would occur if a loop out occurs in the template strand. Mutations in the mutS and mutL mismatch repair genes in E . coli (21) and in theirhomologs MSH2, MLHl, and PMSl in S. cereuisiae (12) render simple repeat sequences unstable. In humans, mutations in the MutS and MutL homologs are associated with hereditary nonpolyposis colorectal cancer (22-251, and tumors in hereditary nonpolyposis colorectal cancer patients exhibit frequent alterations in the length of repeated sequences (7-9). Thus, mismatch repair genes and RAD5 function in opposite ways, respectively,by decreasing and increasing the probability of length alterations of repeat sequences. Cis-acting elements lying within the repeat sequences or in the adjacent regions are involved in the expansion of repeats in fragile X syndrome, myotonic dystrophy,spinocerebellar ataxia type I, and in Huntington's disease (6,261.Trans-acting factors,
however, could also have a role in repeat length alterations in these and otherneurological disorders and in various cancers. It is possible that human counterparts of RAD5 and of the proteins that functionally interact with RADS also contribute to repeat length alterations in human diseases. Acknowledgments-We thank P. M.J. Burgers for the giR of yeast RPA and P.Sung for discussions. REFERENCES 1. 2. 3. 4. 5. 6.
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