THE JOURNAL OF BKXOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 6, Issue of February 25, pp. 3177-3182,199O Printed in U. S. A.

The Primary Structure of the 3%kDa Subunit of Human Replication Protein A*

(Received for publication, September 15, 1989)

Lorne F. Erdile$, Marc S. Wold& and Thomas J. Kelly From the Department of Molecular Biology and Genetics, The Johns Hopkins Medical School, Baltimore, Maryland 21205

Replication protein A (RP-A) is a complex of three polypeptides of molecular mass 70, 32, and 14 kDa, which is absolutely required for simian virus 40 DNA replication in vitro. We have isolated a cDNA coding for the 32-kDa subunit of RP-A. An oligonucleotide probe was constructed based upon a tryptic peptide sequence derived from whole RP-A, and clones were isolated from a Xgtll library containing HeLa cDNA inserts. The amino acid sequence predicted from the cDNA contains the peptide sequence obtained from whole RP-A along with two sequences obtained from tryptic peptides derived from sodium dodecyl sulfate- polyacrylamide gel-purified 32-kDa subunit. The cod- ing sequence predicts a protein of 29,228 daltons, in good agreement with the electrophoretically deter- mined molecular mass of the 32-kDa subunit. No sig- nificant homology was found with any of the sequences in the GenBank data base. The protein predicted from the cDNA has an N-terminal region rich in glycine and serine along with two acidic and two basic segments. Monoclonal antibodies have been raised against the 70- and 32-kDa subunits of RP-A. The cloned cDNA has been overexpressed in bacteria using an inducible T7 expression system. The protein made in bacteria is recognized by a monoclonal antibody that is specific for the 32-kDa subunit of RP-A. This monoclonal an- tibody against the 32-kDa subunit inhibits DNA rep- lication in vitro.

Efforts to understand the replication of chromosomal DNA in animal cells have long been frustrated by the large size and complexity of their genomes. In order to overcome this prob- lem, many investigators have used viral genomes as model systems for studying the processes involved in DNA replica- tion. Simian virus 40 (SV40)’ has been extensively studied as a model system because the replication of its genome shares

* This work was supported by National Institutes of Health Grant GM42780. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 505249.

$ To whom correspondence should be sent. Tel.: 301-955-4190. I Present address: Dept. of Biochemistry, University of Iowa Col-

lege of Medicine, 51 Newton Rd., Iowa City, IA 52242. 1 The abbreviations used are: SV40, simian virus 40; T antigen,

SV40 large tumor antigen; SSB, single-stranded DNA-binding pro- tein; RP-A, replication protein A; SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatograph(y); IPTG, isopropyl- l-thio-P-D-galactoside.

many features with the replication of animal cell chromo- somes (DePamphilis and Wassarman, 1982; Kelly et al., 1988). SV40 DNA replication requires only a single virus-encoded protein, large T antigen, the other replication functions being provided by the host cell.

In order to identify the cellular proteins involved in SV40 DNA replication, our laboratory has used a hypotonic extract of human tissue culture cells in which SV40 DNA is able to replicate in vitro (Li and Kelly, 1984, 1985). DNA replication in vitro shares many characteristics with SV40 DNA replica- tion in uiuo, including a requirement for the SV40 origin of replication, a requirement for large T antigen, and a require- ment that extracts be made from cells that are permissive for viral replication in viuo (Li and Kelly, 1984, 1985; Stillman and Gluzman, 1985; Wobbe et al., 1985). Our laboratory has undertaken fractionation of crude extracts made from HeLa cells and reconstitution of replication activity. Through this approach, we have obtained evidence for the involvement of at least seven cellular factors in the complete replication of SV40 origin-containing DNA (Wold et al., 1989). Five of these have been highly purified: DNA polymerase cu-primase com- plex (Wobbe et al., 1987; Wold et al., 1989); topoisomerases I and II (Yang et al., 1987); proliferating cell nuclear antigen (Prelich et al., 1987; Wold et al., 1988, 1989); replication protein C (the catalytic subunit of protein phosphatase 2A) (Virshup and Kelly, 1989); and replication protein A (RP-A) (Wold and Kelly, 1988; Virshup et al., 1989), also known as replication factor A (Fairman and Stillman, 1988) or HeLa SSB (Ishimi et al., 1988).

RP-A is absolutely required for DNA replication in a recon- stituted system (Wold and Kelly, 1988; Fairman and Stillman, 1988; Ishimi et al., 1988). It is believed that RP-A participates in a very early step in initiation because it is required for T antigen-dependent origin-dependent unwinding of the DNA template (Wold and Kelly, 1988). It is not known whether RP-A participates in the elongation step of replication as well as in initiation. RP-A is purified from HeLa cells as a complex consisting of three subunits of 70,32, and 14 kDa (Wold and Kelly, 1988; Fairman and Stillman, 1988; Ishimi et al., 1988). This complex is very tightly associated since all three subunits cosediment in glycerol gradients run in 6 M urea (Fairman and Stillman, 1988) or in 1.7 M urea with 0.5 M KC1 (Wold and Kelly, 1988). RP-A is an SSB, demonstrating a lOOO-fold greater affinity for single-stranded DNA than for double- stranded DNA as measured by a nitrocellulose filter binding assay in which the binding of radiolabeled denatured DNA was competed with unlabeled single- and double-stranded DNA (Wold et al., 1989). Other investigators have reported that the affinity of RP-A for single-stranded DNA is 30-fold greater than for double-stranded DNA, as determined by measuring the DNA concentration at which 50% of the DNA

3177

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3178 The 32-kDa Subunit of Human Replication Protein A

is bound to a nitrocellulose filter (Fairman and Stillman, 1988). When the subunits of RP-A were separated on an SDS- polyacrylamide gel, transferred to nitrocellulose, and probed with radiolabeled single-stranded DNA, only the 70-kDa sub- unit was detected (Wold et al., 1989), indicating that the single-stranded DNA-binding activity may reside exclusively in this subunit. Other SSBs, including Escherichia coli SSB and the adenovirus DNA-binding protein, are able to substi- tute for RP-A in the initial unwinding step in SV40 DNA replication, but they are unable to substitute for RP-A in the complete DNA replication reaction (Wold et al., 1987; Dean et al., 1987; Virshup and Kelly, 1989). This suggests either that a specific replication complex is formed with RP-A which cannot form with other SSBs or that RP-A has some addi- tional essential activity beyond single-treatment DNA bind- ing. RP-A was assayed for various enzymatic activities in- cluding ATPase, GTPase, 3’ ~5’ exonuclease, endonuclease, helicase, and topoisomerase I; none of these activities was found (Wold et al., 1989).

In order to understand better the role of RP-A in DNA replication as well as the nature of the strong interactions among the three subunits, we have undertaken the isolation and sequencing of the cDNAs coding for the three polypeptide subunits. We report here the cloning of a cDNA coding for the 32-kDa subunit.

MATERIALS AND METHODS

Pur&cation of RP-A-RP-A was purified as described (Wold and Kelly, 1988) except that the 1.3 M KSCN wash from the Affi-Gel Blue column was concentrated and desalted by passing it over a small (0.2 ml) hydroxylapatite column and eluting bound material with buffer F containing 70 mM potassium phosphate (Weld and Kelly, 1988). Peak fractions had a protein concentration as high as 1 mg/ ml.

Peptide Sequencing-Peptide sequence was obtained from both whole RP-A and from the separated subunits. Approximately 1 nmol (150 fig) of RP-A was reduced in 100 mM Tris (pH 8.0), 1% SDS, 20 mM dithiothreitol for 60 min at 60 “C. After cooling to room temper- ature, cysteine residues were carboxymethylated by the addition of 0.05 volume of 0.44 M iodoacetamide (freshly made) with incubation at room temperature in the dark for 30 min. Then 9 volumes of -20 “C ethanol were added along with 3.85 rg (2.5% w/w relative to RP-A) of L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma; prepared from bovine pancreas). The protein was allowed to precipitate overnight at -20 “C and then was collected by centrifugation and resuspended in 0.3 ml of 100 mM NH4HC03 (pH 7.5). An additional 3.85 eg of trypsin was added to make a final ratio of 1 fig of trypsin/20 pg of RP-A, and incubation was carried out for 4 h at 37 “C. Samples were frozen in liquid Nz and stored at -70 “C.

For sequencing the separated subunits, 150 pg was reduced, car- boxymethylated, and ethanol precipitated as above, without trypsin, and then was subjected to SDS-polyacrylamide gel electrophoresis in a Mini Protean II (Hoefer Scientific) using a 10% running gel and a 5% stacking gel (Laemmli, 1970). The protein was then transferred to nitrocellulose, and portions of the filter containing each separated subunit were excised and individually blocked with polyvinylpyrroli- done (Aebersold et al., 1987). Trvnsin freshly made UD in Tris (100 mM, pH 8.2), 5% acetonitrile was then added, and digestion was allowed to proceed overnight at 37 ‘C. The amount of trypsin used was 3.75 pg for the 70-kDa subunit, 1.875 pg for the 32-kDa subunit, and 0.75 gg for the 14-kDa subunit. The supernatant was frozen in liquid N1 and stored at -70 ‘C.

Trypsin-digested whole RP-A was injected onto a Brownlee 30 X 2.1-mm Cq column connected to a Hewlett-Packard 1090 HPLC. Peptides were eluted with a gradient from 0 to 66% acetonitrile. Well resolved peaks were directly sequenced in an Applied Biosystems 470 gas phase sequenator. Other peaks were reapplied to the same HPLC column and eluted with a gradient from 0% acetonitrile, 10 mM triethylammonium acetate, pH 5.5, to 100% acetonitrile, 0 mM trieth- ylammonium acetate. Selected peaks from these runs were lyophilized to dryness three times and then subjected to peptide sequencing.

Trypsin-digested separated subunits were loaded on a Brownlee 210 x 2.1-mm C, column and eluted with a gradient from 0 to 78.4%

acetonitrile in 0.06% trifluoroacetate. Selected peaks were sequenced as described.

Oligonucleotide Synthesis and cDNA Library Screening-A 48- nucleotide-long nondegenerate oligonucleotide was synthesized based upon one of the peptide sequences derived from trypsin-digested whole RP-A as described above. This was used to screen a Xgtll library containing HeLa cDNA inserts (Stratagene; a generous gift of Dr. Hank Ratrie, Johns Hopkins Medical School).-Phage were plated on E. coli strain Y 1090 and grown for 12 h on LB plates containing 50 rg/ml ampicillin. Plaqu& were transferred to nitrocel- lulose filters that were prehybridized in 6 x SSPE, 5 X Denhardt’s solution, 0.1 mg/ml sheared salmon testes DNA (Sigma), 0.1% SDS, and 20% formamide for 2-8 h at 37 “C. The filters were then hybrid- ized overnight at 37 “C in the same solution with oligonucleotide that had been 5’ end labeled with [T-~‘P]ATP using T4 polynucleotide kinase (U. S. Biochemicals). Filters were then-washed twice, with brief shaking, at room temuerature in 6 x SSC. 0.1% SDS. shaken in 6 X SSC, 0.1% SDS at room temperature for 30’min, and then washed once in 0.2 X SSC, 0.1% SDS for 30 min at 42 “C. All positive plaques were purified through two additional rounds.

Subcloning and DNA Sequencing-Human cDNA inserts were excised from Xgtll by cutting with EcoRI and cloned into the EcoRI site in the polylinker of the plasmid pBluescript KS+ (Stratagene, subsequently designated PBS). DNA sequencing was performed by the dideoxv method (Sanger et al.. 1977) using a Seauenase II kit (U. S. Biochemicals) according to the manufactnrer’s mstructions. Se- quencing was done directly on double-stranded plasmids following denaturation by alkali and reannealing at 37 “C as described by Ausubel et al. (1987). Plasmids prepared by the boiling miniprep procedure (Holmes and Quigley, 1981) were extracted twice with phenolchloroform, l:l, and twice with chloroform, ethanol precipi- tated, digested with 1250 units of RNase Tl for 1 h at 37 “C, and extracted and precipitated again prior to sequencing. The ends of the cDNA insert were sequenced using the KS and SK 17-mer primers as well as the Ml3 forward and reverse 20-mer primers (Stratagene). Internal sequences of the cDNA were determined from deletions constructedby digesting with restriction enzymes that cut within the cDNA and within the pBS polylinker. Some regions of the cDNA were sequenced by inserting Sau3Al subfragments into the BamHI site in the pBS polylinker. The recombinantplasmid containing the RP-A 32-kDa cDNA cloned in the EcoRI site of nBS will subseauentlv be referred to as pLE1. The entire cDNA was sequenced on both strands, and most regions were read on at least two separate sequenc- ing gel runs.

Bacterial Expression of Cloned Protein-The cloned gene was overexpressed in E. coli from the bacteriophage T7 $10 promoter in a host in which T7 RNA polymerase is carried on a X-prophage and is expressed from the E. coli lucUV.5 promoter under the inducible control of the 1acZ repressor. The plasmid PET-8c (a gift of Dr. F. W. Studier, Brookhaven National Laboratory), which contains the T7 610 promoter, was cut at the single NcoI site. The 5’ overhang was filled in with Klenow polymerase, and an 8-nucleotide-long BamHI linker (New England BioLabs) was ligated onto the ends. The RP-A 32-kDa cDNA insert was excised from pBS. First, the pLE1 recom- binant plasmid was cut in the polylinker with EcoRV. Second, a BanHI 8-nucleotide linker was ligated onto the EcoRV end. Third, both PET-& and pLE1 were cut with BumHI, and the 4.5-kilobase linearized PET-8c as well as the 1.7-kilobase cDNA fragment from pLE1 were gel purified. These two fragments were ligated together and transformed into E. coli HMS 174. Ampicillin-resistant colonies were selected, and mini-lysate DNA preps indicated that a plasmid containing the cDNA in the correct orientation relative to the T7 promoter had been obtained (designated pLE2). This plasmid was transformed into the E. coli strain BL21 (DL3) in which the T7 RNA polymerase is expressed under the inducible control of the lac repres- sor.

For the induction of a recombinant protein, freshly transformed bacteria were grown overnight in LB medium containing 50 pg/ml ampicillin. The next day, the overnight culture was diluted 1:lOO in M9 medium containing-50 pg/ml ampicillin and grown at 37 “C for anproximately 4 h to an ODW of 0.6. Isopropyl-1-thio&D-galactoside (IPTG) was added to 0.4 mM to induce the T7 polymerase, and incubation was continued. After 30 min, rifampicin was added to a final concentration of 100 fig/ml. Bacteria were harvested 2 h after induction with IPTG by a brief centrifugation and resuspended in l/ 140 volume of 50 mM Tris, 1 mM EDTA (pH 8.0). Cells were frozen and thawed three times, sonicated to break up high molecular mass DNA, and then diluted into SDS-polyacrylamide gel loading buffer

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The 32-kDa Subunit of Human Replication Protein A 3179

(Laemmli, 1970). Samples were boiled for 3 min prior to loading on an SDS-polyacrylamide gel for analysis.

Preparation of Monoclonal Antibodies-Two lo-week-old BALB/c female mice (Jackson Laboratories) were each immunized with 20 pg of RP-A in Freund’s complete adjuvant. The mice were boosted 4 weeks later with 10 fig of RP-A in Freund’s incomplete adjuvant. An additional injection of 10 fig of RP-A in phosphate-buffered saline was done after an additional 2 weeks. Three weeks after that, a final injection of 10 pg of RP-A in phosphate-buffered saline was admin- istered. Fusions were performed 4 days after the final injection. The fusions were screened against RP-A both by enzyme-linked immu- nosorbent assay and by immunoblotting, and positives were cloned twice by limiting dilution. The antibody 71 that recognized the 32- kDa subunit of RP-A was of isotype IgGi.

Zmmunoblotting-Proteins were transferred electrophoretically from SDS-polyacrylamide gels to a nitrocellulose membrane in 0.5 X Laemmli gel buffer, without SDS, but containing 20% methanol. The filter was stained briefly with 0.1% Ponceau S in 3% trichloroacetic acid and then destained with several changes of water. A 1:50-fold dilution of monoclonal antibody 71 in Western blot buffer (0.15 M NaCl, 0.01 M NaP04, pH 7.5, 1 mM EDTA, 0.2% Triton X-100, 1 mM NaN3) containing 4% bovine serum albumin was added, and incuba- tion was carried out for 2 h at room temp. The blot was washed four times with Western blot buffer without bovine serum albumin. In- cubation was then performed for 1 h at room temperature with a second antibody, rabbit anti-mouse IgG (Pel-Freez, affinity purified) at a l:lO,OOO dilution, in Western blot buffer with 4% bovine serum albumin. The filter was washed as before. Finally, the blot was incubated for 90 min at room temperature with ‘Z51-labeled protein A (ICN; >3OCi/pg) diluted l:lO,OOO-fold in Western blot buffer with 4% bovine serum albumin and washed as before.

Inhibition of Replication with Monoclonal Antibodies-Crude HeLa cytoplasmic extract (Li and Kelly, 1984) was incubated, either with buffer H (Wold and Kelly, 1988) containing 15 mM KC1 or with monoclonal antibody in buffer H containing 15 mM KCl, for 15 min at 0 “C, 15 min at 32 “C, and then for 10 min at 0 “C. At this point, buffers, SV40 origin-containing DNA, [32P]dCTP, and T antigen were added to the final concentrations required for in uitro replication as described previously (Wold et al., 1989). Replication was allowed to proceed for 2 h at 37 “C, and samples were treated as described (Wold et al., 1989).

RESULTS

Cloning of a cDNA for the 32-kDa Subunit of RP-A-A 22- amino acid peptide sequence was obtained from a tryptic digest in solution of whole RP-A. That sequence was read as Ser/Gln-Ala-Val-Asp-Phe-Leu-Ser-Asn-Glu-Gly-Ala-Ile- Tyr-Ser-Thr-Val-Asp-Asp-Asp-His-Phe-Lys. Using the table of preferred codon choice for human coding sequences (Lathe, 1985), a single nondegenerate 48-residue-long oligonucleotide of sequence 5’-GCTGTGGACTTCCTGTCCAATGAGGGC- GCCATCTACTCCACAGTGGAC-3’ was synthesized based upon amino acids 2-17. This probe was used to screen a Xgtll HeLa cell cDNA library at low stringency (a final wash at 42 “C! in 0.2 x SSC). Approximately 200,000 plaques were screened, and 21 plaques that hybridized with the probe were plaque purified through three cycles of screening with the oligonucleotide. DNA was prepared from each of these bac- teriophages and digested with EcoRI. The HeLa cDNA inserts released by this digestion were subcloned into the EcoRI site of PBS for further analysis. In order to determine whether any of these cDNAs encoded the amino acid sequence that had been obtained by peptide sequencing, the inserts were subjected to DNA sequencing using primers homologous to the polylinker region of the PBS vector. One of the cDNA inserts was found to be homologous with the oligonucleotide probe at 37 out of 48 positions. Translation of the DNA sequence yielded the amino acid sequence Gln-Ala-Val-Asp- Phe-Leu-Ser-Asn-Glu-Gly-His-Ile-Tyr-Ser-Thr-Val-Asp- Asp-Asp-His-Phe-Lys. This is identical to the amino acid sequence obtained by direct peptide sequencing, except that residue 11 was erroneously determined to be alanine by pep-

tide sequencing rather than histidine as predicted from the cDNA. These 2 residues have very similar retention times (12.02 min for alanine uersus 12.24 min for histidine).

The recombinant plasmid consisting of the cDNA insert described above cloned into the EcoRI site of pBS was desig- nated pLE1. The complete sequence of both strands of the cDNA was determined by using a series of deletions con- structed at unique restriction sites within the insert. The insert was found to contain 1512 base pairs between flanking EcoRI sites (Fig. 1). Within this sequence there is an open reading frame beginning with the first AUG at nucleotide 78 and ending with a TAA at nucleotide 888. No other long open reading frame is found within this cDNA (Fig. 1). The cDNA contains 77 nucleotides of 5’-untranslated sequence and 622 nucleotides of 3’-untranslated sequence. The five nucleotides immediately upstream of the first AUG codon, CCAAG, are a reasonable match for the eucaryotic translation initiation consensus sequence CC(A/G)CC (Kozak, 1984). In particular, the 32-kDa subunit cDNA has an A at position -3, which is the case for 79% of eucaryotic initiator codons.

The derived amino acid sequence strongly suggests that the cloned cDNA codes for the 32-kDa subunit of RP-A. The single long open reading frame of the cDNA specifies a polypeptide 270 amino acids long, including the N-terminal methionine residue. The predicted molecular mass of this protein is 29,228 daltons, in good agreement with the molec- ular mass of 32,000 daltons determined from SDS-polyacryl- amide gel electrophoresis. The open reading frame contains two amino acid sequences that are identical to those derived from sequencing tryptic peptides derived from SDS-polyacryl- amide gel-purified 32-kDa subunit. The peptide sequences Ile- Met-Pro-Leu-Glu-Asp-Met-Asn-Glu-Phe and Ala-Pro-Thr- Asn-Ile-Val-Tyr-Lys were obtained from two different tryptic fragments derived from SDS-polyacrylamide gel-purified 32- kDa subunit. As is shown in Fig. 1, both of these sequences are present in the coding region of the cloned cDNA, and both are preceded by a lysine residue, as would be expected for tryptic peptides. For this reason, along with confirmatory evidence based on a monoclonal antibody that specifically recognizes the 32-kDa subunit (see below), we are confident that we have isolated a cDNA coding for the 32-kDA subunit of RP-A.

Two significant features of the predicted amino acid se- quence were noted. The N terminus of the protein is unusually rich in glycine and serine. Of the first 30 amino acids, 9 (30%) are glycine, and 7 (23%) are serine. Outside of this region, the protein as a whole is not especially glycine- or serine-rich, being 7.4% glycine and 10.4% serine, values that approximate those determined from human protein-coding sequences (Doolittle, 1986). An additional feature of this protein is the presence of several regions of very high acidic or basic char- acter. There are two clusters of basic residues: one between position 37 and position 45 (net charge of +5), and one between amino acids 127 and 145 (net charge of +5). There are two acidic domains: one between residues 95 and 123 (net charge of -7), and one between position 247 and 270 (net charge of -4). Overall, however, the 32-kDa subunit of RP-A contains approximately equal amounts of acidic and basic residues (28 acidic, 30 basic).

The coding region of the 32-kDa subunit cDNA was searched against the GenBank DNA sequence data base ver- sion 61.0, released October 1989 translated into amino acid sequence, as well as against the National Biomedical Research Foundation protein sequence data base version 22.0, released October 1989. No matches of any significance were found. In addition, the protein-coding sequence was examined for zinc

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3180 The 32-kDa Subunit of Human Replication Protein A

CGGCCGCGTTCTGTGGTTTTCCGCTATTCCCCCAOACCCG MTCGTGACCARG

64 77 genes can be expressed from the bacteriophage T7 410 pro-

moter.2 The plasmid is carried in E. coli BL21 (DE3), a protease-deficient strain in which the bacteriophage T7 RNA polymerase is carried on a X-prophage under the control of the lac operon. The orientation of the cloning sites in the vector is such that the T7 TI#I transcription termination sequence lies immediately downstream of the inserted gene. Logarithmically growing bacteria are induced with IPTG, an inducer of the lac operon, resulting in production of the T7 RNA polymerase and transcription of the inserted gene. The T7 expression vector used carries the AUG initiator codon from the T7 gene 10 protein, along with its efficient transla- tion initiation signals, downstream from the phage 410 pro- moter. This AUG codon lies within an NcoI recognition site within the vector PET-8c. This site was converted to a BamHI site into which was ligated the BanHI site of the pLE1, which contains the RP-A 32-kDa subunit cDNA cloned into the polylinker EcoRI site, to produce the expression plasmid pLE2. Due to the way in which pLE2 was constructed, the open reading frame, beginning from the PET-8c AUG codon, contains 105 nucleotides between the PET-8c AUG and the first AUG codon of the cDNA open reading frame. As a result the translation product will consist of an N-terminal fusion of 35 amino acids onto the 270 amino acids of the RP-A 32- kDa subunit. Of these 35 additional amino acids, 10 are derived from the vector, the BamHI linker, and pBluescript polylinker sequences, while 25 are coded for by the 5’-untrans- lated region of the 32-kDa subunit cDNA.

1 11 n * H 9 G F E s Y 0 6 S Y 0 G

ATG TGG UC AGT GGA TTC GM AGC TAT GGC AGC T:C TCA TAC GGG G&A

21 31 AGGYTPSPGGFGSPAF

GCC Got GGC TAC AC0 CAG TCC CCG GGG @SC TTT CGA TCG CCC GCA CCT

41 SQAEKKSRARAQHIVP

XT CM GCC GM AAG AM TCA AGA GCC CGA GCC CAG CAC ATT GTG CCC

51 61 CTISPLLSATl.“DE”F

TGT wr ATA TCT cw cm CTT TCT occ ACT TTG GTT GAT GM GTG TTC

71 A I G N ” E I 6 “TIVGII

AGA ATT GGG MT OTT GAG ATT TCA $3 GTC ACT ATT GTG GGG ATC ATC

61 91 R “A E KA D TII Z "I I[ I DD

AGA CAT GCA GAG AAG ‘XT CCA ACC AX ATT GTT TAC AA.. ATA GAT GAC

101 111 M T A A P ” D” R ‘, WV D T D D

ATG ACA GCT GCA CCC ATG GAC GTT CGC CAG TGG GTT GAC ACA GAT GAC

121 T 6 S E N T Y V P P E T Y” R”

ACC AGC MT GAA MC ACT GTG GTT CCT CCA GAA ACA TAT GTG AAA GTG

131 141 AGHLRSFQNKKSLVAF

GCA CGC CAC CTG AGA TCT TTT CAG AAC AAA lvLG AGC CTG GTA GCC TTT

151 AT Y PI 8 DY I L, T T” IL

AX ATC ATG CCC CTG GAG CAT AT0 MT GAG TTC ACC ACA CAT ATT CTG

161 171 EVINAHHVLSKANSQP

GM GTG ATC AAT GCA CAC ATG GTA CTA AGC AA,, GCC MC AGC CAG CCC

181 191 SAGRAPISNFGMSEAG

TCA OCA GGG AGA GCA CCT ATC AGC AAT CCA GGA ATG AGT GM GCA ~3%

201 N F G G N 6 FM P A N G L T " A

MC TTT GGT GGG AAT AK TTC ATG CCA GCA AAT GGC CTC ACT GTG GCC

211 221 PNPYLNLIKACPRFEG

CAA MC CAG GTG TTG MT TTG ATT AAG GCT TGT CCA AGA CCT GAA GGG

231 Id N F 0 D l. K N P L K" n S" 6

TTG AAC TTT CAG OAT CTC AAG AAC CAG CTG AM CAC ATG TCT GTA TCC

241 251 s I K OA VD rA8 a sea r r

TCA ATC AAG CM GCT GTG GAT TTT CTG AGC MT GAG GGG CAC ATC TAT

261 8TV DD Da FE.3 T D A E

TCT ACT GTG GAT GAT GAC CAT TTT AM TCC ACA GAT GCA GAA TAA

CTGGATCTAACTOGOTACCTGAGATATTTTACAGCTGG 954 AGCTCTGCATATGTCTGGCCAGGGGGC TTCTAGGAAGTAGGTTTCATCTATCAAATGTCTCCTC 101s TGACTTCCTTTTG-CTTACCTGCTCTTCTGTTTTATTTTGTTTTGTTTG~~TCAGAG~AG 1062 ATGGGCAATTGACAGGGATGCAATCCAGGGTGGGATTTCTTGAGGMGTTACAAATAAGCTTGT 1146 TACAACATCMGATAGATGTTGGAAGGATGCT~~CCA~AGAGTACTTACATAGT~TCAGG 1210 AGTTTCTCTTCTT-TGTTTACT~TG-GATGA~AGGACCAG~CGTTAT~~A~ 1274 CCTAOCCAGAMCCTGCTOOCCTCTGCCTOTTTTCATTTCCCACTTT~TTGTGT-ATT~T 1338 TTCAGGMTTOCACTTTCCTGCTTGTCATGACTTTTTGACACACTT~CATGACGTGTGTTTCTG 1402 TGMCATGMGTTCTOCGGTAGTtCCTCCA~CAOAGGIATTT 1466 TGTAC-TAAATACAGTCATATGTTTAATAAAACAGTTCTACCG 1512

125

173

221

269

317

365

413

461

509

557

605

653

701

749

797

815

890

FIG. 1. The sequence of the RP-A 32-kDa subunit cDNA and the predicted amino acid sequence. Nucleotides are num- bered in the 5’ to 3’ direction, beginning and ending with the EcoRI sites used to insert the cDNA in Xgtll. The deduced amino acid sequence is shown immediately above the corresponding nucleotide sequence. The two amino acid sequences in boldface were read from tryptic peptides derived from SDS-polyacrylamide gel-purified RP-A 32-kDa subunit. The amino acid sequence outlined in italics was read from a tryptic peptide of whole RP-A.

fingers (Berg, 1986) and leucine zippers (Landschultz et al., 1988), functional domains found in many DNA-binding pro- teins, as well as for the adenine nucleotide-binding consensus sequence (Walker et al., 1982). None of these was found. We also failed to note any internally repeated domain within this protein.

Expression of the 32-kDa Subunit of RP-A in Bacteria- The cDNA coding for the 32-kDa subunit of RP-A was cloned into PET-8c, a bacterial expression vector in which exogenous

Bacteria containing either pLE2 or the vector PET-8c were induced with IPTG, grown for 2 h at 37 “C, and lysed. The lysates were electrophoresed on SDS-polyacrylamide gels, and the separated proteins were transferred to a nitrocellulose membrane. The filter was probed with a monoclonal antibody, designated 71, which was prepared as described under “Ma- terials and Methods.” When human RP-A is electrophoresed under these conditions, the monoclonal antibody recognizes only the 32-kDa subunit (Fig. 2, lane 1). The principal new protein produced when bacteria pLE2 are induced is a 32-kDa protein that is recognized by monoclonal antibody 71 (Fig. 2, lane 2). We estimate that approximately 2 pg of recombinant protein is produced/ml of cells. This 32-kDa protein recog- nized by the anti-RP-A monoclonal antibody is produced only at very low levels in uninduced cells containing pLE2 (Fig. 2, lane 4) and is not seen in bacteria containing the vector PET- 8c, whether or not they are induced (Fig. 2, lanes 3 and 5). In addition to the predominant 32-kDa product, a slightly smaller species that is also recognized by the monoclonal antibody is produced in considerably smaller amounts upon induction of bacteria containing pLE2. The fact that the cloned cDNA codes for the production of a 32-kDa protein that is recognized by a monoclonal antibody against the 32- kDa subunit of RP-A confirms the identity of the cDNA that we have cloned. It might have been expected-since the bacterial expression construct has an additional 35 amino acids of open reading frame which are not present in the human cDNA-that the bacterially expressed 32-kDa subunit would be somewhat larger than the protein isolated from human cells. It may be that the bacterially expressed protein has undergone some proteolysis in the bacteria or upon cell lysis. It is also possible that the mammalian protein may contain some post-translational modifications, not produced in bacteria, which cause it to run a few kilodaltons larger on SDS-polyacrylamide gels than would be predicted by the size of the cDNA open reading frame. It is apparent from Fig. 2

* Studier, F. W., Rosenberg, A. H., and Dunn, J. J. (1990) Methods Enzymol., in press.

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The 32-kDa Subunit of Human Replication Protein A 3181

4

46-

21.5

14.5

kDa DISCUSSION

FIG. 2. Expression of the RP-A 32-kDa subunit in bacteria. An tmmunoblot of extract from bacteria transformed with TT expres- sion sector pET-SC or vector with the 32-kDa subunit cDNA pLE2 was probed with a monoclonal antibody against the 32.kDa subumt. I,nnc I, 200 ng of RP-A. Lnnes 2 and 4, extract from bacteria transformed with vector contaming the cDNA insert. Lanes 3 and 5, extract from bacteria transformed with vector alone. The extracts m /ones 2 and 9 were prepared from bacteria induced for 2 h with IPTG: those in lanes 4 and 5 were prepared from uninduced cells.

that several proteins present in uninduced bacteria are rec- ognized to some extent by the anti-32-kDa subunit mono- clonal antibody. These species are not detected when the Western blot is probed only with rabbit anti-mouse IgG and protein A without the monoclonal antibody (data not shown). The strongest of these bands runs with a mobility just slightly less than that of the 32.kDa subunit of RP-A; this species may be a bacterial protein that shares some immunological characteristics with the mammalian protein.

A Monoclonal Antibody against the 32.kDa Subunit of RP- A Inhibits DNA Replication in Vitro-In order to examine further the role of the 32-kDa subunit of RP-A in DNA replication zn vitro, we examined whether monoclonal anti- body against the 32.kDa subunit of RP-A would inhibit DNA replication in a cell-free extract. A crude HeLa cytoplasmic was preincubated with either 2.8 or 5.6 Kg of ammonium sulfate-concentrated supernatant from the hybridoma line producing monoclonal antibody 71, and the additional com- ponents required for in uztro replication were then added. Replication was allowed to proceed for 2 h at 37 “C. The products of replication were electrophoresed on an agarose gel and analyzed by autoradiography. The monoclonal anti- body against the 32.kDa subunit of RP-A strongly inhibited DNA replication. Preincubation with 5.6 pg of anti-32-kDa subunit monoclonal antibody produced a 74% inhibition com- pared with preincubation with antibody buffer alone, whereas preincubation with 2.8 pg of antibody resulted in a 72% inhibition of DNA replication (Fig. 3). This inhibition could

a-32 kDs(pg) - - 5.6 2.8 5.6 2.8 add back RP-A - - - - + +

FIG. :3. Effect of anti-32-kDa subunit monoclonal antibody on DNA replication in uitro. Crude HeLa cytosolic extract was incubated as described under “Materials and Methods” either with buffer alone or with monoclonal antibody. The additional components required for in citro DNA replication were added, and replication was carried out for :! h at 3’7 “C. In some cases, purified RP-A was added along with the replication mixture. Products were analyzed by agarose gel electrophoresis and autoradiography.

be completely overcome by the addition of 200 ng of purified RP-A prior to the start of the replication reaction, indicating that the inhibition is due to a specific interaction between the antibody and RP-A and not some nonspecific inhibitory com- ponent in the antibody preparation.

We report the cloning of a cDNA that encodes the 32.kDa subunit of human RP-A, a protein absolutely required for SV40 DNA replication m vitro. The sole long open reading frame within the cDNA would code for a protein of molecular mass 29,228 daltons, a number in good agreement with the molecular mass of the 32-kDa subunit as measured on SDS- polyacrylamide gels. Within the open reading frame are found two peptide sequences identical to those obtained from direct peptide sequencing of the 32-kDa subunit purified by SDS- polyacrylamide gel electrophoresis. The cloned cDNA encodes the production in E. coli of a 32-kDa protein that is recognized by a monoclonal antibody specific for the 32-kDa subunit of RP-A. The predicted amino acid sequence exhibits no signif- icant homologies with any of the proteins in the sequence banks.

One interesting feature of the derived amino acid sequence is the presence of two acidic regions: one of 29 amino acids with a net charge of -7, and one of 24 amino acids with a net charge of -4. Similar acidic stretches are found in several yeast transcriptional activator proteins, including GAL 4 (Laughon and Gesteland, 1984), GCN 4 (Hinnebusch, 1984), and PHO 4 (Legrain et al., 1986). For example, a 19.amino acid region of GCN 4, with a net charge of -5, appears to be sufficient to allow transcriptional activation (Hope and Struhl, 1986). Current models of transcriptional activation propose that the transcriptional activators interact with DNA through a DNA-binding domain and interact with other pro- teins through the acidic activator domain. It is possible that the acidic regions of the 32-kDa subunit of RP-A are involved in protein-protein interactions with the other subunits of RP- A and perhaps with other proteins involved in replication. Now that the cDNA for the 32.kDa subunit is in hand and has been expressed in bacteria, mutagenesis can be under- taken to define the sequences within this protein necessary for the intersubunit interactions as well as interactions with other replication proteins. We have obtained the peptide

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3182 The 32-kDa Subunit of Human Replication Protein A

sequence from the gel-purified 70 and 14-kDa subunits3 and are currently engaged in isolating cDNAs encoding these two proteins.

The exact role of the 32- and 14-kDa subunits of RP-A in DNA replication in vitro is still unknown. We have presented evidence that a monoclonal antibody directed against the 32- kDa subunit is able to inhibit replication, indicating that it does play an essential role in replication. RP-A is required both for complete replication as well as in the presynthetic template-unwinding reaction carried out in the presence of origin-containing DNA and large T antigen. The RP-A com- plex has single-stranded DNA-binding activity that is intrin- sic to the 70-kDa subunit (Wold et al., 1989). The single- stranded DNA-binding activity of the 70-kDa subunit seems to be sufficient for presynthetic template unwinding, since single-stranded binding proteins from autologous sources will substitute for RP-A in this reaction. The single-stranded DNA-binding activity, however, appears not to be sufficient for the complete replication reaction, since SSBs from autolo- gous sources will not substitute for RP-A in replication. It is possible that the 32- and 14-kDa subunits provide some enzymatic activities that are necessary for replication but not for unwinding. Another possibility is that the proteins in- volved in SV40 DNA replication form a large multiprotein complex such as has been proposed to be involved in the replication of E. coli (Baker et al., 1986) and bacteriophage X- DNA (Dodson et al., 1986). There might then be very specific interactions between RP-A and other replication proteins which are unable to be reproduced by heterologous SSBs. The 32- and 14-kDa subunits of RP-A might play an essential role in mediating these protein-protein interactions. The ability to overexpress these proteins and carry out mutagenesis should allow delineation of the precise nature of the interac- tions in which these molecules participate.

Acknowledgments-We would like to thank Clark Riley and Van Vogel of the Biopolymers Laboratory of the Howard Hughes Medical Institute (Johns Hopkins) for very able assistance with peptide se- quencing and Brian Byers of the same laboratory for oligonucleotide synthesis. We would also like to thank Vicki Saylor for production of monoclonal antibodies.

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L F Erdile, M S Wold and T J KellyThe primary structure of the 32-kDa subunit of human replication protein A.

1990, 265:3177-3182.J. Biol. Chem. 

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