Dual roles of a multiprotein complex from S. cerevisiae in transcription and DNA repair

Cell, Vol. 75, 1379-1387,

December

31, 1993. Copyright

0 1993 by Cell Press

Dual Roles of a Multiprotein Complex from S. cerevisiae in Transcription and DNA Repair William J. Feaver; Jesper Q. Svejstrup; Lee Bardwell,t* A. Jane Bardwell,t Stephen Buratowski,§ Keith D. Gulyas,Il Thomas F. Donahue,11 Errol C. Friedberg,t and Roger D. Kornberg’ *Department of Cell Biology Stanford University School of Medicine Stanford, California 94305 tLaboratory of Molecular Pathology Department of Pathology University of Texas Southwestern Medical Center Dallas, Texas 75235 5Whitehead Institute for Biomedical Research Nine Cambridge Center Cambridge, Massachusetts 02142 IlDepartment of Biology Indiana University Bloomington, Indiana 47405

Summary Yeast RNA polymerase II initiation factor b, homolog of human TFIIH, is a protein kinase capable of phosphorylating the C-terminal repeat domain of the polymerase; it possesses a DNA-dependent ATPase activity as well. The 85 kd and 50 kd subunits of factor b are now identified as RAD3 and SSLl proteins, respectively; both are known to be involved in DNA repair. Factor b interacts specifically with another DNA repair protein, SSLS. The ATPase activity of factor b may be due entirely to that associated with a helicase function of RADB. Factor b transcriptional activity was unaffected, however, by amino acid substitution at a consewed residue in the RADB nucleotide-binding domain, suggesting that the ATPaselhelicase function is not required for transcription. These results identify factor b as a core repairosome, which may be responsible for the preferential repair of actively transcribed genes in eukaryotes. Introduction Fractionation of yeast extracts has revealed five RNA polymerase II initiation factors, termed a, b, d, e, and g. All five factors are required in addition to RNA polymerase II for accurate initiation at nine yeast and mammalian promoters tested (Sayre et al., 1992). Cloning, sequencing, and functional studies (Conaway and Conaway, 1993; Feaver et al., unpublished data; Henry and Kornberg, unpublished data) have disclosed the following homologies

SPresent address: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720.

between yeast and mammalian initiation proteins (yeast, rat, and human): a, E, TFIIE; b, 6, BTFP/TFIIH; d, 7, TFIID; e, a, TFIIB; and g, Pr, TFIIF. Factor b is the most complex of the initiation proteins and the only one so far that has been shown to play a catalytic role. Factor b activity in transcription copurifies with five polypeptides (Svejstrup et al., unpublished data) and with two catalytic activities, a protein kinase specific for the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II and a DNA-dependent ATPase (Feaver et al., 1991b). Phosphorylation of the CTD was thought to be essential for the transition from transcription initiation to elongation (Usheva et al., 1992), but both protein kinase activity and the CTD have proven to be dispensable for transcription with purified components (Serizawa et al., 1993; Li and Kornberg, submitted). Factor b is nonetheless required in purified systems, reflecting a crucial role for the DNA-dependent ATPase or for another function. Recently, the gene for the largest subunit of BTF2/TFIIH, the human counterpart of factor b, was reported to be identical with the previously characterized ERCC3 gene (Schaeffer et al., 1993) (now called XPB). Mutation of this gene can result in defective nucleotide excision repair (NER) and is associated with the hereditary diseases xeroderma pigmentosum and Cockayne syndrome (Weeda et al., 1990). A yeast homolog of XPB, called SSLP, was isolated as a suppressor of a stem-loop structure situated upstream of a translation initiation site (Gulyas and Donahue, 1992). This gene has been independently cloned by hybridization with theXP8 gene and designated RAD25 (Park et al., 1992). SSLP is an essential gene (Gulyas and Donahue, 1992), but nonlethal mutant alleles can result in increased sensitivity to ultraviolet (UV) radiation (Guylas and Donahue, 1992; Park et al., 1992) and defective NER in a defined cell-free system (Wang et al., submitted). Several other essential genes are known to be involved in DNA repair in yeast. The RAD3 gene is required for NER and encodes a DNAdependent ATPaselDNA helicase (reviewed by Friedberg, et al., 1991). Like SSLP, RAD3 has a human homolog, designated ERCC2 (XPD), which is also implicated in xeroderma pigmentosum and Cockayne syndrome (reviewed by Hoeijimakers, 1993). The screen for suppressors of defective translation from which SSLP was derived further yielded SSLl (Yoon et al., 1992). Viable mutant ssll alleles confer increased UV sensitivity to yeast (Yoon et al., 1992), suggesting that SSLI, like SSLP and RADS, is involved in DNA repair. We report here that RAD3 and SSLl proteins are the 85 kd and 50 kd subunits of purified factor b. SSL2 protein is not a component of purified factor b, but does interact with it. Our findings strengthen the previously demonstrated connection between transcription and DNA repair (Bohr et al., 1985; Sweder and Hanawalt, 1993,1992; Leadon and Lawrence, 1992) and may have implications for the structure of the mammalian protein G/BTFPTTFIIH.

Cell 1360

A M

b RADB

M

b

b

Figure 1. Comigration During SDS-PAGE and Cross-Reactivity with Anti-RAD3 Antibody of the 65 kd Subunit of Factor b and RAD3 Protein

RAD3

(A) Purified factor b(l.2 pg, lane 2)and purified RAD3 protein (250 ng, lane 3) were analyzed in an SDS-polyacrylamide (10%) gel. Proteins were visualized by staining with Coomassie blue. To better reveal the smaller factor b subunits, 1.6 pg of purified factor b (lane 5) was analyzed as described above: the gel was stained with silver. Molecular weights of the markers (lanes 1 and 4) are given in kilodaltons. (S) Purified factor b (250 ng, lane 1) and purified RAD3 protein (75 ng, lane 2) were fractionated as described in (A), transferred to nitrocellulose, and probed with affinity-purified antiRAD3 antibody. Molecular weights are given in kilodaltons.

llO64-

47-

33-

24-

161

Results Factor b Activity Resides in a Five-Subunit Complex Yeast RNA polymerase II initiation factor b was identified and purified on the basis of its ability to restore transcription activity to a heat-inactivated nuclear extract (Feaver et al., 1991a). Three polypeptides with estimated molecular weights of 85, 75, and 50 kd were found to copurify with transcription activity. Cloning of the gene for the approximately 75 kd subunit (Gileadi et al., 1992), termed TFB7, allowed the development of an improved purification procedure (Svejstrup et al., unpublished data). A six-histidine tag was added to the C-terminus of transcription factor bl (TFBl), allowing purification of factor b to near homogeneity in five steps, including chromatography on Ni2+-nitrilotriacetic acid-agarose (Hochuli et al., 1987). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed five polypeptides, with apparent molecular weights of 85, 75, 55, 50, and 38 kd (Figure lA, lane 5). The three largest polypeptides appeared to correspond to those previously identified as subunits of factor b (Feaver et al., 1991a), and the two smaller polypeptides are components of the factor b complex as well (Svejstrup et al., unpublished data). This subunit composition of factor b more closely resembles those reported for human BTF2/TFIIH and rat liver factor 6. BTF2 copurifies with five polypeptides of apparent molecular weights 90,60,43,41, and 35 kd (Gerard et al., 1991), while the most highly purified 6 fraction contains eight polypeptides with molecular weights of 94, 85, 68, 46, 43, 40, 38, and 35 kd (Conaway et al., 1992). RAD3 Encodes the 85 kd Subunit of Factor b RADB protein purified from an overexpressing strain (Naegeli et al. 1992) comigrated in SDS-PAGE with the 85 kd subunit of factor b (Figure 1 A, lanes 2 and 3). Affinitypurified anti-RAD3 antibody reacted strongly and to a comparable extent with purified RADB protein and with the

2

85 kd polypeptide from an equivalent amount of factor b (Figure 1 B; lanes 1 and 2). lmmunoblotting further showed that RAD3 protein coeluted with transcriptional activity in the final chromatographic step of factor b purification (Figure 2). The DNA-dependent ATPase activity of purified RAD3 protein was closely comparable to that of factor b: 1.2 nmol of ATP hydrolyzed per picomole of RAD3 in 1 hr at 30°C, and 1.4 nmol of ATP hydrolyzed per picomole of factor b (assuming a single copy of all five polypeptides in the factor b complex). Purified RAD3 protein on its own did not possess any detectable factor b activity in the heat-

Fraction 232425292729293031323334353937393940

Transcription

25

26

2728

29

303132

33

34

35

36

37

38

RAD3

11084

-

47WC?Stertl

Figure 2. RAD3 Protein Copurifies with Factor b Activity in Transcription Consecutive fractions from the Mono Q HR 5/5 fast protein liquid chromatography column were assayed for transcription activity (2 PI, top panel) or transferred to nitrocellulose and probed with affinity-purifed anti-RAD3 antibody (50 pl, lower panel). Molecular weights are given in kilodaltons.

Transcription-DNA 1381

Repair Connection

am-

A

IgG -1 1

RAD3

RAD3

Rad3-21

-II 2

4

1

2

4

2500

1

ant+

B

W 1 _

TFBl I I

3 u-l -L-dmn,

1 E

I

In

2

4

Protein

6

I 8

( p g)

Figure 4. Cells Harboring a Mutation in RAW That Eliminates Repair Activity Retain Factor b Transcription Activity

DNA

A Bio-Rex 70fraction was prepared from a wild-typeRAD3 strain (S14; open squares) and from a mutant rad3 strain (S14-21; closed diamonds). Both fractions were assayed for factor b activity in a heattreated nuclear extract (top panel). The amount of protein assayed is given in micrograms. Specific transcripts were quantified on a Radioanalytic Imaging System (Ambis Systems) and the results were plotted (lower panel: transcription (CPM), ordinate axis and amount of protein assayed, abcissa axis). Figure 3. Affinity-Purified Anti-RAD3 Antibody and Affinity-Purified Anti-TFBl Antibody Inhibit Transcription In Vitro (A) Either affinity-purified anti-RAD3 antibody or purified total immunoglobulin G from a nonimmunized rabbit were added to a yeast nuclear extract (120 pg). Reaction mixtures were incubated on ice for 1 hr, after which template, nucleotides, and transcription buffer were added. In vitro transcription was performed as described in Experimental Procedures. The amounts of antibody added are given in micrograms. Specific transcripts (top panel) were quantified on a Phosphorlmager (Molecular Dynamics). Percent of maximal transcription was plotted (lower panel) on the ordinate axis and amount of antibody added was plotted on the abcissa axis (Control immunoglobulin G, open squares; anti-RAD3, closed diamonds). (B) Same as (A) except that the antibody used was affinity-purified anti-TFBl (Control immunoglobulin G, open squares; anti-TFBI, closed diamonds). Quantitation was performed on a Radioanalytic Imaging System (Ambis Systems).

inactivated nuclear extract assay, demonstrating that one or more of the other factor b subunits are required for function in transcription (data not shown). Affinity-purified anti-RAD3 antibody specifically inhibited RNA polymerase II transcription in yeast nuclear extract (Figure 3A). In parallel experiments with affinity-purified anti-TFBl antibody, specific inhibition of transcription was observed in nuclear extract (Figure 38) and also in a system reconstituted from purified factors (data not shown). More anti-RAD3 than anti-TFBl antibody was

required to obtain a comparable level of inhibition. Similarly, the anti-RAD3 antibody immunoprecipitated factor b-SSL2 complex less efficiently than did anti-TFBl antibody (see below). Possibly many RAD3 epitopes are not exposed when the protein is complexed with the other components of factor b. Finally, the identification of RAD3 protein as the 85 kd subunit of factor b was supported by N-terminal sequencing. The various polypeptides of purified factor b were separated by reverse phase high pressure liquid chromatography. The N-terminal sequence found for the isolated 85 kd subunit was M-G-F-X-l-D-X-L-X-V, where X represents an amino acid that could not be determined. This sequence is identical to that deduced from the DNA sequence of the ffAD3 gene (Naumovski et al., 1985), with the exception of the second amino acid, which from the DNA sequence should be lysine rather than glycine. The yields of phenylthiohydantoin amino acid on the second and sixth cycles were low, which may account for the misassignment. RAD3 DNA Repair Activity is Not Essential for Factor b Transcriptional Activity Although RAD3 is an essential gene, nonlethal mutations have been identified that confer markedly increased sensi-

GAL4bd-TFBl GAL4adBSLl

+

AL4bd-TFBl + AL4ad-SNF4

GAL4bd-SSL2 + GAL4adBSLl Figure 5. Association

GAL4bdBSL2 GAL4adBNF4 of TFBl

A

M

b

rSSL1

B

rSSL1

110

-

84

-

+

and SSLI In Vivo

(A) Combinations of GAL4bd and GAL4ad fusion plasmids were transformed into reporter strain Y153 (Durfee et al., 1993). The plasmids carried the GAL4bd fused to TFBI (GAL4bd-TFBl) or to SSL2 (GAL4bd-SSL2) and a GAL4ad fused to SSLI (GAL4ad-SSLl) or to Snf4 (GAL4ad-SNF4). Only the combination of TFBl and SSLl fusions activated transcription at the GAL-/f/S3 gene and thereby allowed growth on selective media. A second reporter gene (GAL-lec2) also was activated only with the combination of TFBl and SSLl fusions (data not shown).

tivity to UV radiation, a condition that is indicative of defective NER (Naumovski et al., 1985). One such mutation, rad3-27, changes a highly conserved lysine residue in the putative nucleotide-binding domain to glutamate. These observations imply a dual role for RAD3: a nonessential function in DNA repair and an additional essential function. Our finding that RAD3 is a component of factor b suggests that the essential function is in RNA polymerase II transcription. If so, then it follows that the DNA repair function of RAD3 is not required for transcription. To test this possibility directly, we prepared factor b fractions from wild-type (514-o) and rad3-27 mutant (S14-21) strains. These fractions were indistinguishable in assays for factor b transcriptional activity (Figure 4). It remains to be shown directlythat therad3-27 mutation abolishes DNA-dependent ATPaselhelicase activity, but if so, it may be concluded that the RAD3 ATPaselhelicase is dispensible for factor b transcription activity. A different mutation in the same conserved lysine residue has been independently shown to inactivate RAD3 ATPaselhelicase activity (Sung et al., 1988). SSLI Encodes the 50 kd Subunit of Factor b The identification of SSLl as a component of factor b was initially suggested by interaction of SSLl with the TFBl subunit of factor b in vivo. The two-hybrid system (Fields and Song, 1989; Chien et al., 1991) was employed with a yeast strain harboring a TF67 to a GAL4 DNA-binding domain (GAL4bd) fusion gene and two reporter genes under GAL control, one expressing HIS3 and the other LacZ. This strain was transformed with plasmid libraries carrying fusions of yeast genomic DNA to the GAL4 activation domain (GAL4ad). Plasmids that could support activated transcription only in combination with the GAL4bd-TFB7 fusion gene (Figure 5) were sequenced from the GAL4ad junction. Three sequences were obtained: one identical to that of SSL7 and two that were not found in current

33 -

24 -

2

Figure 6. Comigration During SDS-PAGE and Cross-Reactivity with Anti-SSLl Antisera of the 50 kd Subunit of Factor b and Recombinant SSLl (A) Factor b (1.6 ug; Mono Q fraction 26, lane 2) and 300 ng of purified recombinant SSLl were analyzed in an SDS-polyacrylamide (10%) gel. Proteins were visualized by silver staining. Molecular weights of the standards (lane 1) are given to the left in kilodaltons. (B) Factor b (lane 1) and recombinant SSLl (lane 2) were transferred to nitrocellulose and probed with anti-SSLl antisera. Molecular weights are given in kilodaltons.

databases. The SSL7 sequence lacked the first 80 amino acids, suggesting that this region of the protein is not required for SSLl-TFBl interaction. Additionally, in studies to be published elsewhere, SSLl protein has been shown to interact with RAD3 protein in vivo and in vitro (Bardwell et al., 1994). SSLl protein purified from bacterial cells comigrated in SDS-PAGE with the 50 kd subunit of factor b (Figure 8A). Equimolar amounts of recombinant SSLl protein and the 50 kd subunit reacted with anti-SSLl antibodies to about the same extent (Figure 8B). The immunoreactive species cofractionated with transcriptional activity in the final step of factor b purification (Figure 7). Finally, a six-residue N-terminal sequence of the 50 kd subunit, (P,V,G,S)-V(V,I)-I-(R,S)-E, was in agreement with that expected for amino acids 2-7 of SSLl (P-V-V-l-S-E). We conclude that the 50 kd subunit of factor b is SSLl protein. SSL2 Is Not a Subunit of Purified Factor b but Interacts with It The recent report that the largest subunit of BTF2ITFIIH is ERCCB (XPB) protein (Schaeffer et al., 1993) prompted us to investigate whether factor b might contain the yeast homolog of this protein, SSLP (Gulyas and Donahue, 1992). None of the five polypeptides of purified factor b reacted with anti-SSLP antisera in Western blots. A trace of a cross-reacting polypeptide was observed in gels (see Figure 8A, lane 2) and blots(data not shown) with an apparent molecular weight of 105,000, which is larger than that expected from the sequence of SSL2 (95 kd), but similar to that found for SSL2 translated in vitro (Figure 8; Figure

Transcription-DNA 1383

Repair Connection

Fraction -

22

23

24

25

26

27

28

29

30

31

32

33

_I .‘,

antisera: mTfbl)Ip[ Factorb: - - + -

+

-

+

200

I’

I**‘* e

97 69

ss12

46 Fraction 22

23

24

25 26

27

28

29

30

31

32

30-

33

84 -

---i-234577

Figure 8. SSLP Protein Coimmunoprecipitates 47 -

33 -

Figure 7. Factor b Activity in Transcription Protein

with Purified Factor b

In vitro translated 35S-labeled SSLP protein was loaded directly on an SDS-polyacrylamide (10%) gel (5 fmol, lane I), or was immunoprecipitated with anti-TFBl (lanes 2 and 3) anti-RADP (lanes 4 and 5) or antiRAD3 antisera (lanes 6 and 7) in the presence (plus sign, lanes 3, 5, and 7) or absence (minus sign, lanes 2, 4, and 6) of 1 pmol of factor b prior to gel analysis. lmmunoprecipitation reactions contained 1 pmol of “S-labeled SSL2 protein and 40 ug of unlabeled reticulocyte lysate proteins. Molecular mass standards are shown on the left. Cofractionates

with SSLl

Consecutive fractions from Mono Cl HR5/5 were assayed for transcrip bon activity (0.1 ul, top panel) or transferred to nitrocellulose and probed with antiSSL1 antisera (20 ul, lower panel). Molecular weights are given in kilodaltons. The amount of SSLl exactly paralled the factor b transcriptional activity.

9). While this minor protein component has yet to be unequivocally identified, we may conclude that SSLP is not a stoichiometric component of purified factor b. Although only a trace contaminant in purified factor b, SSLP was more abundant at earlier stages of the fractionation procedure and coeluted with transcriptional activity during Niz+-nitrilotriacetic acid-agarose and subsequent chromatographic steps (Svejstrup, et al., unpublished data). These observations suggested that SSLP was a component of a larger factor b complex, but that it was less tightly associated than the five subunits of the purified factor and so was gradually lost during fractionation. The possibility of SSLP-factor b interaction was investigated by immunoprecipitation of 35S-labeled SSL2 protein, made by transcription and translation in vitro. The labeled protein was immunoprecipitated by affinity-purified antiRAD3 and anti-TFBl antibodies in a factor b-dependent manner (see Figure 8; Figure 9). The antiRAD3 antibodies gave less precipitation than those against TFBl (see Figure 8) even though amounts of antibodies were used that precipitated the cognate antigens (RAD3 or TFBl proteins translated in vitro) to the same extent. As noted above in regard to the lesser effect of antiRAD3 antibodies on transcription, more RAD3 than TFBl epitopes may be obscured by contact with other components of factor b. Specificity of the immune reaction was demonstrated by a lack of precipitation with antibodies against RAD2 protein, which is neither a component of purified factor b nor is it present in the reaction (see Figure 8, lanes 4 and 5). Specificity of SSL2-factor b interaction was shown by the following: coimmunoprecipitation in the presence of

the approximately lOOO-fold excess of unlabeled proteins of the reticulocyte lysate used for in vitro translation; no detectable coimmunoprecipitation of irrelevant labeled proteins translated in vitro, such as fos (Figure 9, lanes 4 and 5) and jun (data not shown); no detectable coimmunoprecipitation of RAD14 protein present in the same binding reaction as SSL2 (Figure 9); and a dissociation constant of 125 nM, attesting to the strength of the SSL2factor b interaction. Additionally, studies to be reported

labeled z g protein: ur%H Factor b: 200 97 --

4-

fos

+-R14

---m-m-

l

234567

Figure 9. Specificity of SSL2-Factor Not Interact with Factor b

b Interaction: fos and RAD14 Do

In vitro translated %-labeled RAD14, SSLP, and fos proteins were loaded directly on an SDS-polyacrylamide (10%) gel (5 fmol protein/ lane, lanes I-3)orwereimmunoprecipitated withanti-TFBl antiserum (lanes 4-7) in the presence (plus sign, lanes 5 and 7) or absence (minus sign, lanes 4 and 6) of 1 pmol of factor b prior to gel analysis, lmmunoprecipitation reactions contained 1 pmol of “S-labeled fos (lanes 4 and 5) or SSL2 plus RAD14 protein (lanes 6 and 7) and 40 ug of unlabeled reticulocyte lysate proteins. Molecular mass standards are shown on the left.

Cell 1304

elsewhere have demonstrated that in vitro translated SSL2 protein specifically interacts with purified RAD3 protein (Bardwell et al., 1994). Discussion RAD3 and SSLl proteins are identified here as the 85 kd and 50 kd subunits of yeast RNA polymerase II initiation factor b, respectively, on the basis of the following: the recombinant proteins comigrated with the corresponding factor b subunits in SDS-PAGE; the recombinant proteins and factor b subunits reacted comparably with specific antisera; the immunoreactive polypeptides copurified with factor b transcriptional activity; and the N-terminal sequences determined for the two subunits matched those deduced from the DNA sequences of the corresponding genes. Additional evidence for the identification of the 85 kd subunit with RAD3 came from the inhibition of in vitro transcription by anti-RAD3 antibodies. Although not a subunit of purified factor b, SSLP protein interacted with the factor, as shown by coimmunoprecipitation by antisera against both RAD3 and TFBl polypeptides. As mentioned above, there is evidence that mutations in RAD3, SSLl, and SSLP confer defects in DNA repair, and it has been shown that mutations in TFBl can have this consequence as well (Buratowski, unpublished data). Hence, 4 of the 8 associated or interacting factor b polypeptides for which cloned genes are available (RAD3, TFBl, SSLl, and SSL2) prove to be important for DNA repair. Factor b may conceivably function as a core repairosome (Friedberg et al., 1991); the question arises as to why it is essential for transcription. Others have drawn attention to common features of repair and transcription, such as the requirement for DNA duplex melting in both processes (Schaeffer et al., 1993; Buratowski, 1993). A direct role of factor b in duplex melting seems unlikely, since the DNA-dependent ATPasel helicase activity of the purified factor may be entirely attributable to RADS, and evidence is presented here that this function of RAD3 is dispensable for transcription. The possibility remains that SSL2, an essential protein that may possess a DNA-dependent ATPaselhelicase activity based on its amino acid sequence (Guylas and Donahue, 1992) and that is shown here to interact with factor b, is involved in transcription. However, arequirement for SSL2 in transcription has yet to be directly demonstrated. Whatever the essential role of factor b, its presence in or affinity for transcription complexes may underlie the coupling of DNA repair to transcription. Preferential repair of DNA damage in the template strand of actively transcribed genes has been documented in both prokaryotes and eukaryotes (Bohr et al., 1985; Mellon and Hanawalt, 1989). It is thought that such repair may follow polymerase stalling at sites of DNA damage (Hanawalt and Mellon, 1993). Upon stalling at natural pause sites and other barriers to transcription, RNA polymerases back up and cleave some residues from the growing end of the RNA before resuming chain elongation (reviewed by Kassavetis and Geiduschek, 1993). If a similar phenomenon occurs at sites of DNA damage, RAD3 (or SSL2) helicase may un-

wind the region in front of the polymerase, thereby exposing the damaged residue(s) for repair by the rest of the factor b complex and NER apparatus so that upon resuming transcription, the polymerase will be able to traverse the previously damaged region. RAD3 helicase has a 5’-+ polarity with respect to the DNA strand to which it is bound (Sung et al., 1987; Harosh et al., 1989). Hence, if RAD3 helicase is oriented so its direction of movement is the same as that of transcription, it will translocate on the nontemplate strand, which may be important since RAD3 itself becomes arrested at sites of DNA damage (Naegeli et al., 1992). The identification of RAD3 protein as a subunit of factor b raises the question of whether its mammalian homolog ERCCP (XPD) (Weber et al., 1990) is a subunit of 6/BTF2/ TFIIH. This question is particularly acute since the related repair protein ERCC3 (XPB) was identified as the largest subunit of G/BTF2/TFIIH (Schaeffer et al., 1993), but we have shown here that its yeast horn&g SSL2 is not a subunit of purified factor b. Moreover, as mentioned above, it remains to be determined whether the trace of SSL2 in purified factor b preparations is important for their activity in transcription. The question may be asked more generally whether all factor b-associated or factor b-interacting polypeptides are required for both repair and transcription. The genetic evidence that mutations in RAD3, TFBl, SSLl, and SSLP can all impart UV sensitivity and that all four genes are essential for cell viability is indicative of a role in both processes. The results of inhibition with anti-RAD3 and antiTFBl antibodies reported here provide more direct evidence that the essential role of these two proteins is in transcription. The question will be fully answered when factor b is reconstituted from purified components and when the activities of various combinations of polypeptides in NER and transcription are analyzed in vitro.

Experlmental Procedures Plasmid Construction The GAL4bd (amino acids 1-147) was fused to TFE7 as follows. A

BspEl-Xmal fragment from pTFB1 (Gileadi et al., 1992) coding for amino acids 5-642 was inserted in the Xmal site of plasmid pY3 (Sadowski et al., 1992), which expresses Gal4(1-147) from the AD,47 promoter and which carries a TRP7 marker and CEN/autonomously replicating sequences. The resulting plasmid was designated pGAL4-TFBl. A GAL4bd-SSLZ fusion was constructed as follows. A 1.9 kb Hindlll-Pvull fragment containing the promoter and 5’ coding region of SSLPwas cloned into Ml 3mpl6. An oligonucleotide (GATAACAGGGAAmGTCAGAAGG) was used to introduce an EcoRl site (changes are underlined) between codons 45 and 46, and the resulting fragment was reinserted in the SSLP gene. A 3.4 kb EcoRl fragment carrying the SSLP sequence following codon 46 was cloned downstream of the GAL70 promoter and was able to complement an SSL2 deletion. The EcoRl fragment from this GAL-SW? plasmid (~1572) was cloned in frame into plasmid pY2 (Sadowski et al., 1992). A GAL4ad-SNF4 fusion plasmid was a gifi of S. Elledge. The SSL7 open reading frame (Yoon et al., 1992) was amplified by polymerase chain reaction. Ncol and BamHl restriction sites were introduced at the 5’ end and 3’ end of the open reading frame. Six histidine codons were added at the 5’ end. Primer sequences were 5’-ATATGCTAGCCATCACCATCACCATCACACGGACGlTGAAGGCTA-3’ and 5’-ATATGGATCCTTAAGlTAlTACGGGCTT-3’. The NCOI-

Transcription-DNA 1305

Repair Connection

BamHl fragment was cloned into the corresponding

sites of PET-1 Id

tion. Libraries

carrying

fusions between

Saccharomyces

cerevisiae

(Novagenj.

genesand Ike QAL4ad[Chkn etal., 4OOijwere providedby!?. Plelds

Descriptions of plasmids for in vitro transcription of fos, RADS, RAD14, SSLZ, and TFBI are given elsewhere (Bardwell, 1992; Bardwell et al., 1992, 1994).

(State University of New York, Stony Brook) and transformed into strain YSB257. Positive clones were selected as described on media containing 25 m M 3-aminotriazole and confirmed by the secondary f3-galactosidase assay (Durfee et al., 1993). Plasmidswere rescreened to confirm that activation was dependent on both fusion genes, Sequencing was performed from the junction of the GAL4ad and the novel sequence was compared against sequence databases using the BLAST program (Altschul et al., 1990). One of the positive clones was found to be identical to SSL7. The fusion occurred at a Sau3A site (position 860 in the published sequence [Yoon et al., 19921) making isoleucine 80 the first amino acid derived from SSLl.

Protein Purification Factor b tagged at the C-terminus of TFBl with six histidine residues was purified to near homogeneity from yeast whole-cell extracts by chromatography on Bio-Rex 70 (Bio-Rad), Phosphocellulose (Whatman P-l 1). Ni”-nitrilotriacetic acid-agarose (Qiagen), Phenyl Superose HR 5/5 (Pharmacia), and Mono Q HR 5/5 (Pharmacia). Approximately 100 ug of factor b was obtained from 400 g of cells. A more detailed description of the yeast strain and the purification protocol will be published elsewhere (Svejstrup et al., unpublished data). RAD3 protein was purified from an overexpressing strain as described (Nagegeli et al., 1992). The fractions assayed in Figure 4 were derived from strains 514-O (a rad3::H/S3 ade2-101 his3A leu2-1 mef8-7 tfpl ura3-52 [pNF3001]) and S14-21 (a rad3::H/S3 ade2-707 his3A /eu2-1 met8-1 trpl ura3-52 ]pNF3001-211) (Song et al., 1990). Whole-cell extracts from these strains were prepared and chromatographed on BioRex 70 as described (Sayre et al., 1992). Recombinant SSLl was expressed and purified as follows. Cells (BL211DE3, 2 I) harboring the SSLl expression construct were grown at 37OC to an OD 600 value of 0.6 and then were induced with 0.4 m M isopropyl 6-D-thiogalactopyranoside for 3 hr at 30°C. Recombinant SSLl was expressed at a high level but was mostly insoluble. Cells were harvested, resuspended in 40 ml of lysis buffer (20% glycerol, 50 m M Tris-HCI (pH 8.0) 500 m M NaCI, 0.2% Tween-PO,2 m M imidazole), and lysed by sonication. Insoluble material was collected by centrifugation and resuspended in 40 ml of insoluble lysis buffer (8M urea, 100 m M sodium phosphate (pH 8.0) 10 m M Tris, 2 m M imidazole) by stirring for 1 hr at room temperature. Material that remained insoluble was removed by centrifugation, and the supernatant was bound in batch to 5 ml of Ni2+-nitrilotriacetic acid-agarose (Qiagen) for 2 hr at room temperature. The slurry was washed in a column with 25 ml of insoluble lysis buffer (pH 8.0) and 25 ml of insoluble lysis buffer (pH 6.3). Virtually homogeneous recombinant SSLI was eluted with 10 ml of 6 M urea, 100 m M sodium phosphate (pH 6.3) 10 m M Tris, and 250 m M imidazole. The yield was about 4 mgll of starting culture. Transcription and ATPase Assays Factor b activity was assayed by its ability to restore transcription activity to a heat-inactivated nuclear extract (Feaver et al., 199la). Nuclear extracts were prepared from strain BJ926, as previously described (Lue et al, 1991). The template used in all transcription assays consisted of the yeast CYC7 promoter fused to a cassette lacking G (Lue et al., 1989). ATPase assays (20 ~1) contained 500 ng of poly(dU) (Pharmacia), 50 t.rM ATP, 2 uCi of [Y-~~P]ATP (Amersham, >5000 Gil mmol), and the buffer of Harosh et al. (1989). Reactions were performed at 30°C for 1 hr. ATP hydrolysis was monitored by thin layer chromatography on PEI-cellulose as described (Feaver et al., 1991 b). Quantitation was performed on a Phosphorlmager (Molecular Dynamics). Proteln Sequencing Approximately 100 trg of purified factor b was applied to a Vydac C4 reverse phase column (0.21 x 15 cm). Polypeptides where eluted with a O%-100% gradient of acetonitrile in 0.1% trifluoroacetic acid. Fractions containing the 85 kd subunit were identified by SDS-PAGE, concentrated by vacuum centrifugation, and subjected to N-terminal sequencing by the Harvard Microchemistry Facility. For the 50 kd subunit, factor b was fractionated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Immobilon, Millipore), and visualized by staining with Coomassie blue (Matsudaira. 1987). The band corresponding to the 50 kd subunit was excised and subjected to N-terminal sequencing by the Harvard Microchemistry Facility. Two-Hybrid Screen Yeast strain Y153 (Durfee et al., 1993) was transformed with pGAL4TFBl to generate strain YSB257. It was determined that the GAL4-

Transcription, Translation, and lmmunoprecipitation In vitro transcription, translation, partial purification of translation products by ammonium sulfate precipitation, and immunoprecipitation were as described (Bardwell et al., 1992). Other Methods Western blots were performed according to Chasman and Kornberg (1990). The affinity-purified anti-RAD3 antibody (Naumovski and Freidberg, 1988) was used at a final dilution of 111000. The secondary antibody/detection reagent was a goat anti-rabbit alkaline phosphatase conjugate (Bio-Rad). Silver staining was as described (Blum et al., 1987). Affinity-purified anti-TFBl antibody was prepared from crude antisera (Gileadi et al., 1992) by immunoaffinity chromatography on TFBl-glutathione transferase-Sepharose followed by flow through a glutathione transferase-Sepharose column. The TFBl-glutathione transferase fusion protein (Gileadi et al., 1992) and glutathione transferase were coupled to cyanogen bromide-activated Sepharose (Pharmacia) according to the instructions of the manufacturer. Affinity purified RADP antibodies (Nicolet and Freidberg, 1987) were obtained as described. For the generation of anti-SSL2 antibodies, a portion of the SSLP coding region (a Spel-Clal fragment corresponding to amino acids 225-332) was fused to the trpE gene using the pAHT2 vector (Koerner et al., 1991). Escherichia coli carrying this plasmid were induced with indoleacrylic acid, resulting in the overproduction of the fusion polypeptide. This polypeptide was cut out of polyacrylamide gels and used to inoculate rabbits. Polyclonal serum was collected 1 O-l 4 days later. Acknowledgments We thank B. Laneof the Harvard Microchemistry Facility for N-terminal microsequencing and S. Elledge, S. Fields, and Ivan Sadowski for two-hybrid screen reagents. A. J. 8. has previously published under the name A. J. Cooper. W. J. F. was supported by a Medical Research Council of Canada Studentship. J. Q. S. was supported by a fellowship from the Danish Research Council. This work was supported by the National Institutes of Health grants GM36659 to R. D. K, CA12428 to E. C. F, GM46498 to S. B., and GM32263 to T. F. D. References Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D.J. (1990). Basic local alignment search tool. J. Mol. Biol. 275, 403-410. Bardwell, L. (1992). Aspects of the biochemistry of nucleotide excision repairin Saccharomyces cerevisiae. PhD thesis, Stanford University, Stanford, California. Bardwell, L., Bardwell, A. J., Feaver, W. J., Svejstrup, J. Q., Kornberg, R. D., and Friedberg, E. C. (1994). Yeast Rad3 protein binds directly to both SSL2 and SSLI proteins: implications for the structure and function of transcription/repair factor b. Proc. Nab Acad. Sci. USA, in press. Bardwell, L., Cooper, A. J., and Friedberg. E. C. (1992). Stable and specific association between the yeast recombination and repair proteins RADl and RADlO in vitro. Mol. Cell. Biol. 72, 3041-3049. Blum, H., Beier, H., and Gross, H. J. (1987). Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93-99.

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DNA

Usheva, A., Maldonado, E., Goldring, A., Lu, H., Houbavi, C., Reinberg, D., and Aloni, Y. (1992). Specific interaction between the nonphosphorylated form of RNA polymerase II and the TATA-binding protein. Cell 69, 871-881. Weber, C. A., Salazar, E. P., Stewart, S. A., and Thompson, L. H. (1990). ERCC2: cDNA cloning and molecular characterization of a human nucleotide excision repair gene with high homology to yeast RAD3. EMBO J. 9, 1437-1447. Weeda, G., van Ham, R. C. A., Vermeulen, W., Bootsma, D., van der Eb, A. J., and Hoeijmakers, J. H. J. (1999). A presumed DNA helicase encoded by ERCC-3 is involved in the human repair disorders xeroderma pigmentosum and Cockayne’s syndrome. Cell 62, 777-791. Yoon, H., Miller, S. P., Pabich, E. K., and Donahue, T. F. (1992). SSL7, a suppressor of a HIS4 5’-UTR stem-loop mutation, is essential for translation initiation and affects UV resistance in yeast. Genes Dev. - _.__ _.--

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Notes Added

Repair Connection

in Proof

The data referred to throughout as Li and Kornberg, submitted, is now in press: Li, Y., and Kornberg, R. D. (1994). Interplay of positive and negative effecters in the function of the C-terminal repeat domain of RNA polymerase II. Proc. Natl. Acad. Sci. USA. We have found that purified SSLP protein can stimulate a reconstituted yeast RNA polymerase II transcription reaction. Others have reported that mutations in the SSLP gene can result in defects in transcription in vivo (Qui, H., Park, E., Prakash, L., and Prakash, S. [1993]. The Saccharomyces cerevisiee DNA repair gene RAD25 is required for transcription by RNA polymerase II. Genes Dev. 7, 2161-2171).