Kenneth R. Williams, and William H. Konigsberg. From the Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven, ...
Vol. 264, No. 19, Iesue of July 5, pp. 10943-10953,1989 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.
The 44P Subunit of the T4 DNA Polymerase Accessory Protein Complex Catalyzes ATP Hydrolysis* (Received for publication, December 19,1988)
John Rush$, Tsung-Chung L h , Michael Quinones& Eleanor K. Spicer, Iris Douglas, Kenneth R. Williams, and William H. Konigsberg From the Department of Molecular Biophysics and Biochemistry, Yale School of Medicine, New Haven,Connecticut 06510
The genes encoding all three T4 DNA polymerase sociating from primer-template junctions (2,8). accessory proteins havebeen cloned into overespresTwo ofthe accessory proteins, 44P and 62P, form a tightly sion plasmids. Induction ofcells harboring these plas- associated complex, which is disrupted only by exposure to mids results in thesynthesis of each accessory protein protein denaturing reagents such as guanidine HCl and SDS’ at levels that approach 10%of the total cellular pro- (9). The subunit stoichiometry (44P:62P) of this accessory tein. The solubility of the accessory proteins after in- protein complex is uncertain, having been evaluated as 4:2 duction at 42 “C ranges from about 60% to greater (lo), 5 or 6 1 ( l l ) and , 3.6 (-+0.6):1 (12). The complex has a than 95%. low level DNA-dependent ATPase activity that isthought to A plasmid thatallows overexpressionof the 44P/62P provide energy for the assembly and maintenance of a replicomplex has been manipulated further to overexpress selectively the 44P subunit without62P,permitting us cation apparatus which consists entirely of T4-encoded replication proteins (13,14).Accessory protein stimulationof the to assess how each subunit contributes to the properties of the 44P/62P complex. A comparison of 44P and enzymatic activities of T4 DNA polymerase requires ATP hydrolysis, not just accessory protein binding, because they 44P(62P by conventionalhydrodynamictechniques shows that44P forms a subcomplex nearlyas large as do not function when non-hydrolyzable analogues are substithe 44P/62P complex. In addition, 44P catalyzesDNA- tuted for ATP (15). Although the third accessory protein 45P has no known dependent ATP hydrolysis with a specific activity similar to that of the 44P/62P ATPase. However, unlike enzymatic activity of its own, it greatly stimulates the 44P/ the44P162Pcomplex,theATPase activity of 44P 62P ATPase (13, 14). Recent Studies suggest that 45P can alone is only slightly stimulated by45P. This suggests stimulate the 44P/62P ATPase in the absence of DNA and that one role of the62P subunit is to facilitate a pro- that DNA and 45P together stimulate this ATPase synergisductive interactionof 44P and 45P. tically (14). Conventional hydrodynamic techniques demonstrate that 45P is a dimeric protein (16). Its ability to form dimers may reflect the coupling of replication complexes on the leading and lagging strands of a replication fork (2). In Genes 44,45, and 62 of bacteriophage T4 encode three DNA addition, 45P is essential for transcription during the late polymerase accessory proteins that areindispensable for viral stages of infection (17) and interactstightly with immobilized replication (1). These three proteins stimulate the enzymatic Escherichia coli RNA polymerase purified from T4-infected activities of T4 DNA polymerase specifically, without exerting cells, but not with unmodified RNA polymerase holoenzyme effects on other DNA polymerases in in vitro DNA synthesis or core (18). Because of its dual role, it has been suggested assays (2, 3). The accessory proteins increase the rates and that 45P may coordinate replication and late transcription processivities of DNA polymerization and 3‘ to 5’ exonucleo- during infection (19). lytic degradation catalyzed by T4 DNA polymerase (4-6). In There are several functional similarities between the auxaddition, they reduce the occurrence of template-switching iliary factors (8, y, and 6) of E. coZi DNA polymerase I11 and and template-slippage errors (7), thereby increasing the ac- the accessory proteins of T4 DNA polymerase (2,3,20). First, curacy of DNA synthesis. These effects appear to arise from both auxiliary factors and accessory proteins extend the enan enhanced affinity of T4 DNA polymerase for the primer- zymatic capabilities of their cognate polymerases without template junction in the presence of accessory proteins. For eliciting new enzymatic activities. For example, these proteins this reason, the accessory proteinsarethought to form a enable their polymerases to use primer-templates that the “sliding clamp” that prevents T4 DNA polymerase from dis- polymerases would not otherwise be able to use efficiently (long single-stranded templates for DNA polymerase I11 and * This work wassupported by National Institutes of Health Grants nicked duplexes for T4 DNA polymerase). In addition, these GM30191 (to E. K. S.) and GM12601-20 (to W. H. K.). The costs of publication of this article were defrayed in part by the payment of proteins greatly increase the processivity of DNA synthesis page charges. This article must therefore be hereby marked “adver- catalyzed by their cognate polymerases, from about 10 to tisernent” in accordance with 18 U.S.C.Section 1734 solelyto indicate several thousand nucleotides incorporated per binding event. this fact. Finally, both sets of proteins “activate” their respective poThe nucleotide sequencefs)reported in thispaper hu.s been submitted lymerases through DNA-dependent ATP hydrolysis (21-23). to theGenBankTM/EMBLDataBankwith accession numberfs) Because of these functional similarities to auxiliary factors, M10160,JV2510,and X00769. $Current address and to whom correspondence should be sent: the accessory proteins can beviewed as loosely associated Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. § Current address: Dept. of Surgery, Brigham and Women’s Hospital, Boston, MA 02115.
The abbreviations used are: SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography; Tricine, N-[2-hydroxy-l,1bis(hydroxymethyl)ethyl]glycine.
10943
10944
T4 DNA
Polymerase Accessogv Protein 44P
subunits of a multi-subunit T4 DNA polymerase holoenzyme, although there is currently no physical evidence for holoenzyme assembly. The nucleotide sequences of genes 44 (12) and 45 (24) were previously determined in our laboratory, and the deduced primary structures of these accessory proteins were confirmed by protein chemical analysis. In this paper, we report the nucleotide sequence of gene 62 and the construction of plasmids that allow overexpression of the accessory proteins. In addition, by reconstructing one of the recombinant plasmids we have been able to assess the contributions of each subunit of the 44P/62P complex. EXPERIMENTALPROCEDURES
Materials-Restriction endonucleases were purchased from New England Biolabs, Boehringer Mannheim, orBethesda Research Laboratories. The BamHI linker (5'-CCGGATCCGG-3'), Klenow fragment, and polynucleotide kinase were purchased from Bethesda Research Laboratories. Linkers were phosphorylated with polynucleotide kinase beforeuse. DEAE-modified membranes, NA-45,were purchased from Schleicher & Schuell and were used according to the instructions of the manufacturer. VAT media consists of 10 g of tryptone, 4 g of yeast extract, 10 g of K2HP04, and1 g of KHzPOl per liter. 44P/62P and 45P purified from T4-infected cells (11) were the generous gift of Nancy Nossal (NIH). Single-stranded DNAcelIulose was prepared from singlestranded calf thymus DNA and Whatman C F l l cellulose according to the method of Alberts and Herrick (25). Hydroxylapatite was purchased from Bio-Rad. Phosphocellulose (P11)was purchased from Whatman and was pre-cycled with acid and base. Proper pretreatment of phosphocellulose was necessary to ensure good separation and high capacity during chromatography. Bacterial Strains and Cloning Vectors--E. coli strain HBlOl [F-, hsdS20 ( a , mg), ara-14, proA2, InCYl, galK2, rpsL20 (Sm'), xyE-5, mtl-I, supE44, recA13, X-] was used for maintainingall plasmid constructions. E. coli JM103 [A(&, pro), thi, strA, supE, endA, sbcBl5, F' traD36, proAB, lacZQZAM15](26), M13 derivatives mp7, mp8, and mp9 (26), and theplasmid vector pUC9 (26) were purchased from Bethesda Research Laboratories. The recombinant plasmid pMU232 was the generous gift of J. Karam (Medical University of South Carolina); pARlOl has been described previously (24). DNA Sequencing-Nucleotide sequencing was performed by the method of Sanger et al. (27), using recombinant M13 with T4 DNA inserts. Growth of M13 on E. coli JM103, preparation of singlestranded DNA templates, and the dideoxy chain terminating reactions were carried out as described previously (12). Sequencing reactions were analyzed on 6 or 8% polyacrylamide gels containing 7 M urea, which were dried under vacuum before autoradiography on Du Pant Cronex 4 film. Cloning Procedures-Restriction fragments, purified by electrophoresis on agarose gels, were adsorbed to DEAE membranes (28) and eluted with buffers containing high concentrations of salt. Ligations and cell transformations were performed according to standard protocols (29). Induction of Cells That Overexmess the Accessory Proteins-Large quantitiesof'induced cells were prepared for purification of the accessory proteins by growth in a 15-liter Microferm fermentor (New Brunswick Scientific) in VAT media supplemented with glucose (10 g/liter) and ampicillin (100 mg/liter) immediately before use. Cells were grown at 30 "C to an Aswof about 0.75 and then induced by adding boiling VAT media until the culture temperature reached 42 "C. After 2 h under inducing conditions, cells were harvested by centrifugation in a Beckman JAlO rotor at 8000 rpm for 10 min a t 4 "C. Induced cells were stored at -70 "C. Purification of 45P, 44P/62P, and 44P from Induced celk-45P was purified according to the method of Nossal (11) from induced cells harboring the plasmid pTL45W, which contains gene 45but not genes 44 or 62 (Fig. 3, panel ZI). Induced cells harboring plasmid pTL151WX (containing genes 45, 44, and 62) or pTL89W (containing genes 45 and 44) were used as starting material for the purification of 44P/62P or 44P, respectively (Fig. 3, panel IV). 44P/62P and 44P were purified according to the method of Morris et al. ( X ) , except cells were lysed by treatment with lysozyme, EDTA, and deoxycholate (30). As described by Morris et al. (16), contaminating 45P was quantitatively retained on DEAE-
Catalyzes ATP Hydrolysis
cellulose along with most other cellular proteins, but 44P/62P was not adsorbed. Unlike the 44P/62P complex, the 44P subunit was weakly retained on DEAE-cellulose. Accordingly, 44P was recovered by developing the column with a linear gradient consisting of buffer DO(40 mM Tris-HC1, pH 7.4,lmM MgCL, 10mM 8-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 12.5% glycerol) and buffer Do + 0.4 M NaCl. Protein concentrations were determined after acid hydrolysis by amino acid analysis using a Beckman 121M amino acid analyzer. SDS-polyacrylamide gel electrophoresis was performed according to the method of Laemmli (31). Protein Chemical Analysis of 62P-For sequence and composition analysis, 62P was purified as a complex with 44P from T4-infected cells (11) and was provided byN. Nossal (NIH).To determine internal amino acid sequences, approximately 400pgof 62P was digested with trypsin at a 441 (w/w) substrate/enzyme ratio for 6 h at 37 "C, and the resulting peptides were separated by reverse phase HPLC asdescribed previously (12). The amino-terminal sequence of intact 62P was determined by automated Edman degradation of 4.2 mg ofcarboxamidomethylated 44P/62P complex on a Beckman model 89OC liquid-phase sequencer. Phenylthiohydantoin derivatives were identified either by amino acid analysis after hydrolysis with hydriodic acid for 18 h a t 130 'C or when necessary by HPLC. Based on the relative yield of phenylthiohydantoin derivatives for cycles 24, the molar ratio of 44P:62P in the complex was 3.6 (-+0.6):1(12). This relatively large ratio permitted unambiguous assignment of the derivatives present a t each cycle of sequencing to either 44P or 62P. Analytical Gel Filtration-The apparent molecular weights of the accessory proteins were determined by analytical gel filtration on a 1.6 X 85-cm column of Sephacryl S300 superfine (Pharmacia LKB Biotechnology Inc.), thoroughly equilibrated with buffer G (150 mM K2HP04,pH 7.0, 1 mM 8-mercaptoethanol, 5 mM MgSO,, 10% glycerol). The column was calibrated with protein standards (high molecular weight kit; Sigma), blue dextran (for the void volume, VO), and deoxyadenosine (for the total volume, V,) dissolved in buffer G. Accessory proteins were dialyzed against buffer G before chromatography. All samples were applied to thecolumn in a total volume of 2 ml and included 1 mM deoxyadenosine as a marker for V,. The hydrostatic pressure on the column was held constant a t a flow rate of 17 ml/h, and 1-ml fractions were collected and monitored for A z ~ . 44P/62P, 44P, and 45P eluted from the column a t concentrations of about 50 pg/ml. Assignments of absorbance peaks in the chromatograms to a particular proteinwere confirmed by SDS-polyacrylamide gel electrophoresis. Fractional retention K, was calculated according to the formula K, = (V, - Vo)/(Vt - Vo),where V. is the peak elution volume for each protein. Sedimentation Velocity Centrifugation-Accessory proteins were examined by sedimentation velocity centrifugation to correct for the contribution of molecular shape to apparent molecular weights, permitting the calculation of true molecular weights (32). Gradients of 5 to 20% sucrose in buffer S (150 mM KzHPO,, pH 7.0, 1 mM 8mercaptoethanol, 5 mM MgSOJ were formed in 14 X 89-mm tubes (Beckman Ultra-Clear). A linear gradient mixing device with a sixport outlet enabled six identical gradients to be cast simultaneously. Protein standards (high molecular weight kit; Sigma), dissolved in buffer S, were gently layered on top of the gradients, which were then spun in an SW41 rotor for 22 h a t 35,000 rpm and 4 "C. Fractions (400 pl) were collected from the bottom of the tubes with a needlepuncture device connected to a peristaltic pump and were monitored for AZ1,. Accessory proteins, dialyzed against buffer S, were cosedimented with carbonic anhydrase(44P/62P),apoferritin (44P), or thyroglobulin (45P), and their concentrations after centrifugation ranged from about 20 to 75 pg/ml. True molecular weights were calculated from empirically determined apparent molecular weights (Mr,app) and sedimentation coefficients ( s ) according to the relationship (10)
Mrmmple = M
- p zM rL z S,,ple
r std
SStd
spp a t d
where std indicates values for the reference standards. ATPase Assays-ATPase assays were performed essentially according to themethod of Piperno et al. (13), except that the standard concentration of 44P or 44P/62P was 7.8 pg/ml, and the standard concentration of 45P was 13.9 pg/ml. [y3'P]rATP (HPLC purified, in Tricine; Amersham Corp.) was used as a substrate and was separated from the product 32Pi by adsorption to Norit A charcoal (Fisher),
T4 DNA Polymerase Accessory Protein 44P CatalytesATP H'mlysis
10945
FIG. 1. Restriction map of recombinant phage Xt306-17. This A-T4 recombinant (34) contains a 5.6kilobase (kb) EcoRI fragment of T4 DNA. The third line shows an expanded map of the EcoRI-Hind111fragment that contains all of gene 62 and regA and part of genes 44 and 43. The direction of transcription of all four T4 genes is from right to left. Arrowsbelow the restriction maps indicate the direction and length of sequence determined from M13 subclones carrying fragments of gene 62.
3 450
V ~ l e I l e L y S ~-1I" l Ia"Vnl """~alylsprglyrc.""""""""~""~~"
541 T l 7 1 " (181)
wvaL.Ss1-
y lL y E
630
157
FIG. 2. Nucleotide sequence of gene 62 and predicted amino acid sequence of 62P.The regions of the sequence verified by peptide sequencing and by matching tryptic peptide compositions are shown by solid and by broken underlining, respectively. The start codon of the regA gene and the stop codon of gene 44 are indicated by overlining. prepared according to themethod of Zimmerman and Kornberg (33). A standard assay mixture contained 10 mM Tris-HC1, pH 7.4,25 mM KC], 5 mM MgCI,, 1 mM dithiothreitol, 400 pg/ml bovine serum albumin, 35 pg/ml sonicated single-stranded calf thymus DNA, and 250 p~ rATP (British Drug House). [ys2P]rATPwas included in the reaction mixtures a t a concentration of 2.25 pCi/mI. Typical assays were performed by preincubating 45 p1 of the assay mixtures without rATP at 37 "C for 5 min, followed by addition of 5 11of 2.5 mM rATP to start the reaction. At appropriate intervals, 25pl aliquots of the assay mixtures were pipetted into 250 pl of 0.1 M HCI and left on ice until all time points had been gathered. Bovine serum albumin (400 pg in 100 p l ) was then added to each acidified sample, followed by250 pl of Norit A suspension. Samples were incubated on ice for 5 min with intermittent shaking. Norit A was then removed by centrifugation in a microcentrifuge for 5 min. The amount of radioactivity remaining in the supernatantswas measured
in a Searle Mark I1 scintillation counter after transferring 500-pl aliquots of the supernatants to10 ml of Optifluor scintillation fluid. Aliquots of the reaction mixtures not taken through acidification and absorption were also removed to measure the total radioactivity in each reaction mixture. Results are presented as the percentage of input radioactivity converted to nonadsorbable radioactivity. Although the [y3'P]rATP used as substrate had been purified by HPLC, the background level of radioactivity in the assay usually corresponded to 1.5 to 3.0% of the input, asignificant amount relative to thefraction of radioactivity converted to nonadsorbable counts by the accessory proteins. For this reason, controls that contained all components of the assay except 44P or 44P/62P were prepared, and the background of nonadsorbable radioactivity was subtracted from values obtained in the presence of 44P or 44P/62P. This allowed sensitive and reproducible measurement of ATP hydrolysis, despite the low signal to noise ratio. ATPase activity was a linear function
T4 DNA Polymerase Accessory Protein 44P Catalyzes ATP Hydrolysis
10946
pARlOl
regA
N43
62
R X A
P
H
I
1
I
28.95
30.1 1
k
pMU232
/
1.
rpbA
45
H
pMU232 c44
I
30.95
32.56
4-
PAR101
'
r-7 LJ
AmP4uc9
PAR101
ti
-lA h
m
f
l
P45
pTL9W
B
Nucleotides
pTL45W Xb
45 Sst I Sph I fragment from M13mp19 I
FIG. 3. Construction of plasmids that allow overexpression of the T4 DNA polymerase accessory proteins. Restriction sites enclosed in brackets were removed during the cloning procedures. N62 and C62 refer to theamino- and carboxyl-terminal coding regions of gene 62. Note that the pTL plasmid numbers correspond to the numeric sum of the T4 gene numbers (hence pTL89 contains genes 44 and 45). Panel I, restriction map of genes 44,45, and62. Ng43 represents the 5' end of gene 43. RpbA, which is upstream of gene 45, encodes an RNA polymerase binding protein (50, 51). The direction of transcription for all the genes shown is from right to left. Restriction sites are EcoRI (R), XhoI (X), AuaI (A), PstI ( P ) ,and HindIII ( H ) , and the numbers below certain sites indicate T4 map units (52). Fragments of this region of the T4 genome already cloned on plasmids pMU232 (53) and pARlOl (24) are indicated. Panel ZI, construction of a plasmid that allows overexpression of gene 45. A HindIII (H) fragment containing gene 45 was removedfrom pARlOl (a pBR322-gene 45 recombinant) and inserted at the Hind111 site of pUC9 to give the plasmid p45 (arrow A). Thesmaller EcoRI-BamHI (R-B)fragment of p45
T4 DNA
Polymerase Accessory Protein 44P Catalyzes
94 kd 67 kd-
43 kd-
30 kd -
20.1kd -
FIG.4. Overexpression of 45P. Total lysates of the host cell without (2nd lane) and with (3rd lane) the plasmid vector pUC9 were made by boiling cells in sample loading buffer (31). Induced cells harboring pTL45W were lysedby sonication and separated into pellet (4th lane) and supernatant (5th lane) fractions by centrifugation. 45P purified from T4-infected cells was run as a standard (6th lane). All samples were electrophoresed on an 11%SDS-polyacrylamidegel. of time for a t least 30 min under standard conditions, except for assays that contained both 44P/62P and 45P, which were linear for only 5 to 7.5 min. RESULTS
The Nucleotide Sequence of Gene 62-The T4 genes 44,45, and 62, encoding the DNA polymerase accessory proteins, map within a 5.6-kilobase region of the T4 genome that also contains the DNA polymerase gene (gene 43) and the regA gene. A restriction fragment spanning much of this region has been cloned previously into phage X (34) (Fig. 1). We have subcloned fragments of gene 62 from X806-17 into M13mp7 and mp8 for nucleotide sequence analysis. The strategy used to obtain the entire gene 62 sequence is summarized in Fig.
ATP Hydrolysis
10947
1, and the nucleotide sequence of the gene and its flanking regions are given in Fig. 2, along with the deduced amino acid sequence of 62P. To identify the initiation codon and correct reading frame for gene 62, the amino-terminal sequence of 62P was determined. The 44P/62P complex, purified from T4-infected cells, was carboxamidomethylated and thensubjected to automatic sequence analysis, as described previously (12). The 3.61 molar ratio of 44P to 62P in the complex and the known sequence of 44P (12) permitted unambiguous assignment of the two amino acids present at each sequencing cycle to either 44P or 62P. In this manner, the sequence of 62P at residues 2-4, 6-11, and 13-16 was determined. This established that synthesis of 62P initiates at thefirst ATG codondownstream from the gene 44 termination codon and that the aminoterminal residue of 62P is serine and not methionine. The nucleotide sequence of gene 62 was verified by demonstrating that it was consistent with the amino acid compositions of randomly selected tryptic peptides of 62P. The 44P/62P complex was disrupted with denaturing reagents, alkylated, and theindividual subunits purified by gelfiltration in 6 M guanidine hydrochloride (12). An examination of the sequence of 62P revealed 27 potential tryptic cleavage sites, of which 24 would be expected to be cleavedefficiently based on the known specificity of trypsin (35), to generate 25 major peptides. After purification by reverse-phase HPLC, the amino acid composition of 12 of these peptides was determined, and an additional two peptides were sequenced, as summarized in Fig. 2. The compositions and sequences of all peptides examined could be predicted from the nucleotide sequence of gene 62, and theconfirmed regions, representing 57% of 62P, were evenly distributed throughoutthe polypeptide. From these results, we conclude that 62P is composed of 187 amino acid residues, with a M,of 21,364.62P contains 24 acidic and 27 basic residues and has a predicted isoelectric point of 9.7. The secondary structure of 62P was predicted by computer analysis (36) using the method of Chou and Fasman (37). According to thisprediction, 62P should contain a relatively high proportion of a-helix (44%) and a low amount of @-strands(9.6%), and the a-helices should be evenly distributed throughout 62P. 62P contains a fairly high percentage of leucine residues (11%) compared with E. coli proteins (7.8%) (38), and these are located predominantly in the carboxyl-terminal third of the protein. Similarly, leucine residues are clustered in the carboxyl-terminal third of 44P (12). However, the significance of this asymmetric distribution of leucine residues is not known. Overexpression of the T4 DNA Polymerase Accessory Proteins-Three plasmids were used to overexpress the accessory proteins 45P and 44P/62P and theaccessory protein subunit
was replaced with an EcoRI-BamHI fragment containing the XPL promotor and cIa7 gene, which had been previously cloned as pTL9W (54), generating pTL45W (arrow B ) . Panel III, construction of a plasmid that site upstream of gene 45in pARlOl contains all three accessory protein genes and theregA gene. The HindIII (H) was changed to a BglII-compatibleBamHI site (B9)by HindIII digestion (partial) and ligation to BamHI linkers, producing the plasmid pARlOlB (arrow A). An EcoRI-Hind111 ( R - H )fragment containing the 3' end of gene 44, gene 62, regA,and the5' end of gene 43 was transferred from pMU232to pARlOlB to give p151 (arrow B ) . Panel IV, construction of plasmids that allow overexpression of genes 44, 45, and 62. The XhoI-BglII (X-Bg) fragment of p151 was inserted between the SaA (S)(XhoI-compatible) and BamHI ( B ) (BgAI-compatible)sites of pTLSW, to produce pTL151W (arrow A). pTL151W was linearized by AuaI ( A ) digestion, resected with Ba131 exonuclease to inactivate regA, and re-ligated, producing pTL151WX (arrow B ) . pTL151W was also digested with PstI (P) and re-circularized to remove regA and the 3' end of gene 62, to generate pTL89W (arrow C ) . To produce a deletion within gene 44, the SstI-SphI fragment of pTL151WX containing gene 44 was replaced with a smaller SstI-SphI fragment from M13mp19 (spanning nucleotides 6241 and 6279 and encoding GTRGSSRVDLQAC), generating pTL107WX (arrow D).
T4 DNA Polymerase Accessory Protein 44P Catalyzes ATP Hydrolysis
10948
94 kd
-
67 kd -
43 kd
-
94 kd
-
67 kd
-
43 kd -
- 44P 30 kd
-
-45P
-44P
30 kd -45 P
-62P 20.1 kd -
14.4 kd -
FIG.5. Purification of overexpressed 44P/62P. Fractions from a preparation of 44P/62P purified from induced cells harboring pTL151WX were electrophoresed on a 15%SDS-polyacrylamide gel. The volumes loaded ontothe gel were in proportion to the total volume of each fraction, so that recovery can be estimated by scanning across the gel. The fractions analyzed were the cell lysate pellet (2nd lane) and supernatant (3rd lane),the DEAE-cellulose pool (4th lane), the hydroxylapatite pool (5th lane), and the phosphocellulose pool (6th lune).
20.1 kd -
14.4kd
-
FIG. 6. Purification of overexpressed 44P. Fractions from a preparation of 44P purified from induced cells harboring pTL89W were analyzed by SDS-polyacrylamide gel electrophoresis, as described in the legend to Fig. 5. The volume of the phosphocellulose pool in the 7th lune is not in proportion to the remaining lanes; more protein was run to facilitate estimation of purity.
45P was co-expressed with the less soluble proteins 44P/62P and 44P. Expression of 44P without the 62P subunit did not 44P in E. coli (Fig. 3). All plasmids contained the accessory markedly alter the solubility of the 44P subunit, which was protein genes downstream of the X P L promotor, and expres- only slightly less soluble than the overexpressed 44P/62P sion of the cloned genes was under the control of the temper- complex (Figs. 5 and 7). ature-inactivatable ~ 1 %repressor, ' whose gene was present on Several attempts were made to construct plasmids for overeach plasmid. The overexpression of the accessory proteins expression of the 62P subunit without 44P. For example, required the removal of the adjacent regA gene, which re- plasmid pTL151W was digested with H i d 1 1 and religated to presses the expression of these and several other T4genes a t remove rpbA, gene 45, and the 5' end of gene 44 (see Fig. 3, the translational level (39, 40). panel I). When cells harboring this plasmid were induced, Cells transformed with plasmid pTL151WX, containing all overexpression of 62P could not be detected by Coomassie three accessory protein genes (Fig. 3, panel ZV), were ther- Blue staining of total cell lysates electrophoresed on SDSmally induced to provide starting material for the purification polyacrylamide gels. This result supports the proposal (41) of 44P/62P. The plasmid pTL89W is similar to pTL151WX that translation of gene 62 is obligately coupled to gene 44 but contains a substantial deletion at the 3' end of gene 62, translation. A deletion was subsequently made within gene so that itdirects the synthesis of only the 44P subunit of the 44,in which 103 codons of gene 44 were replaced with a 44P/62P complex, without producing 62P (Fig. 3, panel ZV). fragment having only 13 codons, to generate pTL107WX (Fig. The construction of pTL89W allowed purification of the 44P 3, panel ZV). This fragment maintained the gene 44 reading subunit, which could not be readily obtained from T4-infected frame and thuspresumably allowed for normal translation of cells. Although plasmids pTL151WX and pTL89W also direct gene 62. In this case, the level of 62P produced was reduced the expression of 45P, induced cells harboring pTL45W ov- at least 10-fold relative to the level obtained with plasmid erexpressed only 45P (Fig. 3,panel ZZ) and were thus preferred pTL151WX, suggesting that 62P may be more sensitive to as startingmaterial for 45P purification. degradation by cellular proteases when overexpressed without Rapidly shifting the temperature of cultures harboringeach intact 44P. of these plasmids from 30 to 42 "C resulted in the efficient Purification of Overexpressed Accessory Proteins-Overexsynthesis of the accessory proteins in a predominantly soluble pressed 45P was purified according to the method of Nossal form. Each overexpressed protein (45P, 44P/62P, and 44P) ( l l ) , which was developedfor purifying 45P from T4-infected accounted for about 10% of the total cellular protein after cells. Lipids, nucleic acids, and most contaminating proteins induction. About 95% of overexpressed 45P was reproducibly were removed from the cell lysate supernatant by precipitafound in the cell lysate supernatant (Figs. 4-6), even when tion with polyethylene glycol. Single-stranded DNA cellulose,
10949
T4 DNA Polymerase Accessory Protein 44PCatalyzes ATP Hydrolysis
15
100
0
“
-
cn
10
5
0
I
I
I
10
20
X,
fraction
B.
A.
FIG. 7. Analytical gel filtration and sedimentation velocity centrifugation of 44P/62P, 44P, and 45P. Panel A shows the selectivity curve obtained after gel filtration of protein standards (0)along with the elution positions of 44P/62P (U), 44P (O),and 45P (m). The protein standards were thyroglobulin (669 kDa), apoferritin (443 kDa), @-amylase(200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa). Protein standard molecularweights were taken from the manual supplied with ~ , of ~protein standards (0)to their the protein standard kit (Sigma). Panel B shows the curve relating the s ~values position in the ultracentrifuge tube along with the sedimentation positions of 44P/62P (U),44P (O),and 45P (m). Fraction 1 is the first fraction collected from the bottom of the ultracentrifuge tube. The protein standards were thyroglobulin (19.2 S),apoferritin (17.6S), @-amylase(8.9 S), alcohol dehydrogenase (7.6 S), and carbonic anhydrase (2.8 S).Sedimentation coefficients for the standards are from Ref. 55.
TABLE I Quaternary structure of overexpressed accessory proteins True molecular weights were calculated from apparent molecular weights and sedimentation coefficients according to the formula given under “Sedimentation Velocity Centrifugation.” Apparent Sedimentation M, coefficient 10-3
45P 44P 35.4 44P/62P
81.6 165108 209
S 2.8 5.4 6.7
TrueM, 10-3
44.2 145
$:E
Subunits
10-~
24.7 35.4/21.4
1.8 3.0 3.0/1.8
which does not adsorb 45P, was then used to remove contaminating DNA binding proteins. A final step of adsorption chromatography on hydroxylapatite provided 45P sufficiently pure (295%)for enzymatic assays. The entireprocedure could be completed in about 3 days. The 44P/62P complex waspurified by the method of Morris et al.. (16). DEAE-cellulose chromatography, which takes advantage of the relatively high isoelectric point of the 44P/ 62P complex (91, was the most effective step in thepurification procedure. Since most proteins have slightly acidic isoelectric points (42), DEAE-cellulose chromatography of the cell lysate supernatant separated44P/62P from most contaminating proteins, including 45P, and simultaneously removed nucleic acids. Subsequent chromatography on hydroxylapatite and phosphocellulose gave nearly homogenous 44P/62P (Fig. 5). Since the 44P/62P complex contains more 44P than 62P
subunits (lO-lZ), it was anticipated that the 44P subunit could be purified by the same procedure used to purify the complex. In fact, this method did allow purification of the 44P subunit to near-homogeneity (Fig. 6). However,while 44P/62P eluted from DEAE-cellulose in the breakthrough and wash fractions, 44P was reproducibly retained. Nevertheless, with steep salt gradients, 44P eluted early in the chromatogram, well separated from most cellular proteins and from 45P. The accessory proteins have been previously purified from cells infected with a T4 regA mutant. Since the expression of accessory protein genes is controlled at thetranslational level by regA protein (39, 40), these mutants overexpress the accessory proteins to as great an extent as currently possible through conventional genetic manipulation of T4. However, use of cells harboring the overexpression plasmids resulted in considerably higher production, which allowedpurification of much larger amounts of the accessory proteins than could be obtained from cells infected with T4 regA mutants. 45P and 44P/62P were purified in 56- and 18-fold greater amounts, respectively, with yields of about 4 mg/gof induced cells. Moreover, the 44P subunit, which had never before been isolated without 62P, was purified at levels that approach 2.5 mg of 44P/g of induced cells. Physical Properties of 44P”Because genes 44 and 62 are adjacent on the 7’4 genome and because the genes are transcribed from the same promotor (43), it is tempting to speculate that proper formation of an active 44P/62P complex
T4 DNA Polymerase Accessory Protein 44P Catalyzes
10950
lo]
T
A T P Hydrolysis
+
I44P/62P
044P/62P 45P 044P/62P A44P 45P
+
I I
+ 45P
0 44P/62P
800
m44P+45P
m44P
m a
U
e.
8 -
600
6-
G 3
400
0
2. 3
4-
I
200 2(61.5)
(18.8)
1
I
0
0
10
0
20
B.
time (rnins)
+
FIG. 8. ATPase assays of 44P/62P and 44P 46P. Time course assays were conducted under standard conditions (Panel A ) , as described under “Experimental Procedures.”Both 44P/62P and 44P were assayed in the presence and in the absence of 45P. Panel B is a histograph of the rates observed in Panel A under the four reaction conditions. The symbols used in the graph are defined in the inset.
20
-
0
15-
lo-
0
5-
/A-A-~
A
O r ‘
0
A.
15 5
10
4 5 concentration ~ OLg/rni)
20
0
B.
5
10
4 5 concentration ~ (pg/rnl)
FIG. 9. Titration of the ATPase activities of 44P/62P and 44P with 45P. 44P/62P (closed circles) and 44P (closed triangles) were both assayed at two concentrations, 23 pg/ml (Panel A ) and 7.8 pg/ml (Panel B ) , while the 45P concentration was varied overan 8-fold range. Reactions were stopped 4 min after adding [y3*P]rATP.
requires co-expression of the genes. For this reason, we won- weights of 44P and 44P/62P were 165,000 and 209,000, redered if the 44P subunit might be monomeric when produced spectively, while the apparent molecular weight of 45P was in theabsence of 62P. Accordingly, the quaternary structures 81,600 (Fig.7A). Control experiments indicated that inclusion of the 44P subunit and the 44P/62P complex werecompared. of deoxyadenosine didnot affect theapparent molecular 45P was included as a reference protein, since it is known to weights of these proteins (data notshown). A second hydrodynamic method, sedimentation velocity be a dimer (10). The accessory proteins and the 44P subunit were subjected centrifugation, also demonstrated that 44P forms a subcomto gel filtration to determine theirapparent molecular plex which is nearly as large as the 44P/62P complex. Each weights. A Sephacryl S300 column was calibrated with high accessory protein was co-sedimented with one of six different molecular weight protein standards which bracketed the ap- standards to generate a calibration line relating sedimentation parent molecular weights anticipated for the accessory pro- coefficients to position in theultracentrifuge tube. The accesteins, and the 44P subunit, the 44P/62P complex, and 45P sory proteins and the standards were paired in such a way were chromatographed individually, with deoxyadenosine as that they would be well separated after ultracentrifugation. a marker for the total column volume, V,. The results, sum- The 44P subunit andthe 44P/62P complex behaved similarly, marized in Fig. 7A, showed that 44P formed a complex nearly with s20,w values of 5.4 and 6.7, respectively, while 45P sedias large as the 44P/62P complex. The apparent molecular mented with a coefficient of 2.8 (Fig. 7B).
T4 DNA
Polymerase Accessory Protein 44P
The apparent molecular weights, obtained by gel filtration, and the sedimentation coefficients allow the calculation of the true, shape-independent molecular weights of the accessory proteins (32). Thesecalculations show that 45P contains 1.8 subunits (TableI), inagreement with previous results that show that 45P is a dimer (10). Furthermore, the 44P subunit has a true molecular weight of 108,000, corresponding to 3.0 subunit molecular weights, and the 44P/62P complex has a molecular weight of 145,000. This suggests that the complex contains 3 subunits of 44P and 1.8 subunits of 62P and is consistent with a stoichiometry of 3:2. Enzymatic Activities of 44P”The enzymatic properties of the 44P subunit and the 44P/62P complex werecompared by assaying each for single-stranded DNA-dependent ATPase activity. 44P and 44P/62P were assayed at equivalent mass concentrations under the assumption that theATPase activity of the subunit, if present, and that of the complex would exhibit similar kinetic requirements. These assays revealed that the 44P subunit was, in fact, enzymatically active, with a specific activity nearly twice that of the complex at equivalent mass concentrations (Fig. 8). However, the ratio of specific activities per 44P subunit is 1.3 (44P44P/62P), if 44P is a trimer andthe stoichiometry of the 44P/62P complex is 3:2 (44P:62P), as suggested above. When assays were repeated in the absence of single-stranded DNA, no ATP hydrolysis was detected, demonstrating that the 44P subunit is a DNA-dependent ATPase, like 44P/62P (data not shown). Previous studies have shown that the third accessory protein 45P greatly increases the activity of the 44P/62P ATPase (13, 14). Accordingly, the ATPase activities of the subunit and the complex were measured in the presence of 45P to determine if stimulation could occur in the absence of the 62P subunit. Although 45P increased the ATPase activity of the 44P/62P complex by a factor of 44, the ATPase activity of the 44P subunit was stimulated only 1.8-fold by 45P (Fig. 8). No ATP hydrolysis was detected when the 44P/62P complex or the 44P subunit was omitted from reaction mixtures, verifying that 45P was free of contaminating ATPase activities. Furthermore, under similar conditions, the experimentally determined rates of ATP hydrolysis catalyzed by all three overexpressed accessory proteins (6.6 nmol/ml/min) were similar to values previously reported for accessory proteins purified from T4-infected cells (5.5 nmol/ml/min), and, in both cases, 45P stimulated the 44P/62P ATPase about 40fold (13). The 44P-45P interaction was examined further by titrating the ATPase activity of the 44P subunit with 45P. The 44P ATPase became more active with increasing 45P concentration, establishing unequivocally that thetwo proteins interact directly (Fig. 9). However, there were striking differences in the levels of ATPase activity attainable by 44P 45P and by 44P/62P + 45P. The plateaus in ATPase activity indicated that (i) theelevated levels of ATP hydrolysis are not caused by increasing the concentration of an ATPase contaminating 45P; (ii) the interaction of 45P with the 44P subunit is not as productive as the interaction of 45P with the 44P/62P complex; and (iii) most importantly, the difference in the specific 45P and44P/62P 45P persists at activities of 44P saturating concentrations of 45P.
+
+
+
DISCUSSION
Expression of Gene 62-As shown in Fig. 2, there is only one nucleotide between the stop codon of gene 44 and the gene 62 start codon. The gene organization at the 3’ end of gene 62 is the same: a single nucleotide separates gene 62 from the start of the regA structural gene. The region imme-
Catalyzes ATP Hydrolysis
10951
diately upstream of gene 62 does notcontain nucleotide sequences usually associated with T4 early or middle promoters (44,45). Thiswould suggestthat gene 62 is transcribed as partof a polycistronic mRNA originating from the middle promotor preceding gene44 (12, 41). In fact, Bowles and Karam (43) have shown that two amber mutants of gene 44 exhibit polar effects on the synthesis of gene 62, whereas a gene 45 amber mutation did not affect synthesis of 44P and 62P. More recently, Gerald and Karam (46) reported that mRNA for 44P is separable from gene 45 mRNA. The mRNA sequence preceding gene 62 contains a potentially weak ribosome binding site (41). Presumably, translation of gene 62 is enhancedby being coupled to translation of gene 44. Trojanowska et al. (41) have postulated that thegene &gene 62 mRNA containsa stem structurethat would sequester the gene 62 start codon and that ribosomes translating the gene 44 mRNA would disrupt the hairpin, enabling reinitiation at gene 62. The results of our efforts to overproduce 62P in the absence of 44P support the conclusion that translation of gene 62in theabsence of gene 44 translation is inefficient. Synthesis of gene 62 protein is known to be regulated at the translational level by regA protein. This regulation probably occurs throughinteraction of regA protein with the translation initiation region of gene 44. If ribosomes do not efficiently initiatetranslation at the gene 62AUG codon independent of gene 44 translation, then only a single regA recognition site would be required for repression of both gene 44 and 62. Roles of the Subunits of the 44P/62P ATPase-The enzymatic activities of the 44P subunit andthe 44P/62P complex were compared to determine the contribution that each subunit makes to thefunctional propertiesof the 44P/62P complex. In principle, this issue could be addressed by separating the subunits of the 44P/62P complex under dissociating conditions andassaying the separated subunitsfor ATPase activity. Because the subunits of the 44P/62P complex are very tightly associated, denaturing reagents such as guanidine HCl and SDS are required for their separation (9). However, the use of denaturing reagents complicates the interpretation of negative results, since the absence of enzymatic activity can then be attributed to incomplete or incorrect refolding of the subunits upon removal of the denaturing reagent. Accordingly, a different approach was employed to assess the role of each of the subunits of the 44P/62P complex. Rather than attempting to dissociate the subunits of preformed complexes, a plasmid that allows overexpression of the complex was manipulated so that each subunit could be overexpressed separately. A plasmid that directed the synthesis of large amounts of soluble 44P was readily constructed. However, ourattempts to construct plasmids that would overexpress soluble 62P without the 44P subunit were not successful. Thus, while the contribution of the 44P subunit to the properties of the 44P/62P complex could be tested directly, the role of the 62P subunit had to be inferred by comparing the properties of the 44P/62P complex and the 44P subunit. Enzymatic assays revealed that the 44P subunit possesses a DNA-dependent ATPase activity, as does the 44P/62P complex. This observation supports the results of a primary structural comparison. Walker et al. (47) found that a number of ATP-binding proteins contained at least one of two conserved sequences, which they speculated might play a role in binding ATP. One of these sequences was found in 44P, but neither conserved sequence was found in 62P (12), which is consistent with the catalytic role of the 44P subunit. Intrigu-
10952
T4 DNA Polymerase Accessory Protein 44PCatalyzes ATP Hydrolysis
ingly, the y and r subunits of E. coli DNA polymerase 111, which are both encoded by the dnaZX gene (48, 49), contain the same putative ATP-binding consensus sequence that is found in 44P. Furthermore,although there is no obvious sequence homology between 62P and the dnaZX gene products, it is interesting that there is similarity with respect to their genetic organization: while y and are encoded by the same gene (48,49), genes 44 and 62 are separated by only one nucleotide. The ATPase activity of the 44P subunit was only slightly stimulated by 45P, in contrast to theactivity of the 44P/62P complex, which was greatly stimulated by 45P (Figs. 8 and 9). A direct interaction of 45P with 44P was indicated by the rise and plateau of the 44P ATPase activity observed as the concentration of 45P was gradually increased. Thus, 44P is stimulated by interacting with 45P, but optimal stimulation requires the presence of the 62Psubunit. An indication thatthe44Psubunit might bea DNAdependent ATPase was provided by earlier studies on the 44P/62P complex. Mace and Alberts (14) demonstrated the physical association of 44P and 62P by sedimenting the complex on sucrose gradients and by assaying the resulting fractions for ATPase activity. Aproteinwith the subunit molecular weight of 44P sedimented slightly slower than the 44P/62P complex and hadDNA-dependent ATPase activity, but was not stimulated by 45P. The studies described here on the enzymatic and physical properties of overexpressed 44P strongly suggest that the slower sedimenting peak observed by Mace and Alberts did, in fact, contain only 44P. In addition, our results suggest that theratio of 44P to 62P subunits in the complex may have been previously overestimated. The data of Mace and Alberts (14), described above, strongly suggest that the 44P/62P preparations used for earlier estimates of subunit stoichiometry contained 44P subunits no longer associated with 62P subunits. Since DEAEcellulose separates 44P/62P from 44P and since Mace and Alberts used DEAE-cellulose chromatography to obtain their preparation of 44P/62P, free 44P subunits could have arisen from selective degradation of 62P during storage or from dissociation of the 44P/62P complex during purificationafter DEAE-cellulose chromatography. This implies that 44P/62P preparations could have been contaminated by varying amounts of free 44P subunits, which could account for the different stoichiometries obtained for the complex as well as for the overestimation of the ratio of 44P to 62P subunits. The subunit stoichiometry has been estimated previously by conventional hydrodynamic techniques (lo), by the relative intensity of Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis ( l l ) , and by liquid phase sequencing of the complex directly, without prior separation of the subunits (12). Among these methods, only sedimentation velocity centrifugation has the potential to detect contamination by free 44P subunits. In summary, the properties of the T4 DNA polymerase accessory proteins are now better understood at the level of subunit functions. 44P is clearly the catalytically active subunit of the 44P/62P ATPase. It binds DNA, binds and hydrolyzes ATP, and interacts directly with 45P. In addition, 44P self-assembles as a large subcomplex, suggesting that it may drive the nucleation of the 44P/62P complex in uiuo. 62P apparently becomes indispensable for DNA replication by modulating the 44P-45P interaction. In this way, the three accessory proteins together generate a potent ATPase activity, which might then become capable of providing the energy that is thought tobe required for the assembly of the T4DNA polymerase holoenzyme at replication forks.
Acknowledgments-We thank Stephen Chin-Bow for his technical assistance during the construction of some of the overexpression plasmids and William E. Balch for his assistance and advice during the sedimentation velocity centrifugation experiments. REFERENCES 1. Epstein, R. H., Bolle, A., Steinberg, C., Kellenberger, E., Boy de la Tour, E., Chevalley, R., Edgar, R., Sussman, M., Denhardt, C., and Lielausis, I. (1964) Cold Spring Harbor Symp. Qunnt. Biol. 28,375-392 2. Nossal, N.G., and Alberts, B. M. (1983) in Bacteriophage T4 (Matthews, C. K., Kutter, E. M., Mosig, G., and Berget, P. B., eds) pp. 71-81, American Society for Microbiology, Wash., D. C. 3. Alberts, B. M., Barry, J., Bedinger, P., Burke, R. L., Hibner, U., Liu, C.-C., and Sheridan, R. (1980) in Mechanistic Studies of DNA Replication and Genetic Recombination (Alberta, B., and Fox, C. F., eds) pp. 449-471, Academic Press, New York 4. Mace, D. C., and Alberts, B. M. (1984) J. Mol. Bwl. 1 7 7 , 313327 5. Newport, J. W., Kowalczykowski,S. C., Lonberg, N., Paul, L. S., and von Hippel, P. H. (1980) in Mechanistic Studies of DNA Replication and Genetic Recombination (Alberts, B., and Fox, C. F., eds) pp. 485-505, Academic Press, New York 6. Venkatesan, M., and Nossal, N. G. (1982) J. Bwl. Chem. 2 5 7 , 12435-12443 7. Nossal, N. G., and Peterfin, B. M. (1979) J. Bioi. Chem. 254, 6032-6037 8. Alberts, B. M., Barry, J., Bedinger, P., Formosa, T., Jongeneel, C.V., and Kreuzer, K.N. (1983) Cold Spring Harbor Symp. Qunnt. Biol. 47,655-668 9. Barry, J., and Alberts, B. (1972) Proc. Natl. Acad. Sci. U. S. A. 69,2717-2721 10. Barry, J., Hama-Inaba, H., Moran, L., Alberts, B., and Wiberg, J. (1973) in DNA Synthesis in Vitro (Wells, R. D., and Inman, R. B., eds) pp. 195-214, University Park Press, Baltimore 11. Nossal, N.G . (1979) J. Bwl. Chem. 2 5 4 , 6026-6031 12. Spicer, E. K., Nossal, N. G., and Williams, K. R. (1984) J. Biol. Chem. 259,15425-15432 13. Piperno. J. R., Kallen, R. G., and Alberts, B. (1978) J. Biol. Chem. 253,5180-5185 14. Mace, D. C., and Alberts, B. M. (1984) J. Mol. Biol. 1 7 7 , 279293 15. Huang, C. C., Hearst, J. E., and Alberts, B. M. (1981) J. B i d . Chem. 256,4087-4094 16. Morris, C.F., Hama-Inaba, H., Mace, D., Sinha, N.K., and Alberts, B. (1979) J. Bwl. Chem. 254,6787-6796 17. Wu, R., Geiduschek, E. P., and Cascino, A. (1975) J. Mol. Biol. 96,539-562 18. Ratner, D. (1974) J. Mol. Biol. 88, 373-383 19. Rabussay, D., and Guideschek, E. P. (1977) in Comprehensive Virology (Fraenkel-Conrat, H., and Wagner, R., eds) Vol. 8, pp. 1-196, Plenum Press, New York 20. McHenry, C. S. (1988) Annu. Reu. Biochem. 59,519-550 21. Wickner, S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73,3511-3515 22. Johanson, K. O., and McHenry, C. S. (1982) J. Bioi. C h m . 267, 12310-12315 23. Burgers. P. M. J., and Kornberg, A. (1982) J. Biol. Chem. 2 5 7 , 11474-11478 24. Spicer, E. K., Noble, J. A., Nossal, N. G., Konigsberg, W. H., and Williams, K. R. (1982) J. Biol. Chem. 257,8972-8979 25. Alberts, B., and Herrick, G. (1971) Methods Enzymol. 2 1 , 198217 26. Messing, J. (1983) Methods Enzymol. 1 0 1 , 20-78 27. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,5463-5467 28. Dretzen, G., Bellard, M., Sassone-Corsi, P., and Chambon, P. (1981) Anal. Biochem. 112,295-298 29. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 30. Mott, J. E., Grant, R. A., Ho, Y.-S., and Platt, T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,88-92 31. Laemmli, U. K. (1970) Nature 227,680-685 32. Siegel, L. M., and Monty, K. J. (1966) Biochim. Biophys. Acta 112,346-362
T4 DNA Polymerase Accessory Protein 44P 33. Zimmerman, S. B., and Kornberg, A. (1961) J. Biol. Chem. 2 3 6 , 1480-1486 34. Wilson, G.G., Tanyashin, V. I., and Murray, N. E. (1977) Mol. Gen. Genet. 166,203-214 35. Allen, G. (1981) Sequencing of Peptides and Proteins, pp. 54-55, Elsevier Science Publishing Co., Inc., New York 36. Cohen, F. (1979) Ph.D. Thesis, Yale University 37. Chou, P.Y.,and Fasman, G. D. (1978) Adv. Enzymol. 4 7 , 45148 38. Lehninger, A. L. (1975) Biochemistry, 2nd Ed., Worth, New York 39. Wiberg, J. S.,Mendelsohn, S., Warner, V., Hercules, K., Aldrich, C., and Munroe, L. (1973) J. Virol. 12,775-792 40. Wiberg, J. S., and Karam, J. D. (1983) in Bacteriophage T4 (Matthews, C. K., Kutter, E. M., Mosig, G., and Berget, P. B., eds) pp. 193-201, American Society for Microbiology, Wash., D. C. 41. Trojanowska, M., Miller, E. S.,Karam, J., Stromo, G., and Gold, L. (1984) Nucleic Acids Res. 12,5979-5993 42. Gianazza, E., and Righetti, P.G. (1980) J. Chromatogr. 193, 1-
8 43. Bowles, M.,and Karam, J. (1979) Virology 9 4 , 204-207 44. Brody, E.,Rabussay, D., and Hall, D. H.(1983) in Bacteriophage T4 (Matthews, C. K., Kutter, E. M., Mosig, G., and Berget, P.
45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55.
Catalyzes ATP Hydrolysis
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B., eds) pp. 174-183, American Society for Microbiology, Wash., D. C. Guild, N., Gayle, M., Sweeney, R., Walker, T., Modeer, T.,and Gold, L. (1988) J. MOLBWL 199,241-258 Gerald, W. L., and Karam, J. D. (1984) Genetics 1 0 7 , 5 3 7 6 4 9 Walker, J. E.,Saraste, M., Runswick, M.J., and Gay, N.J. (1982) EMBO J. 1,945-951 Yin, K.-C., Blinkowa, A., and Walker, J. R. (1986) Nucleic Acids Res. 14,6541-6549 Flower, A. M., and McHenry, C. S. (1986) Nucleic Acids Res. 14, 8091-8101 Williams, K. P.,Kassavetis, G. A., Esch, F. S., and Guiduechek, E. P. (1987) J. V i d . 61,597-599 Hsu, T., Wei, R. X., Dawson, M., and Karam, J. D. (1987) J. Virol. 6 1 , 366-374 Kutter, E., and Ruger, W. (1983) in BacterWphqe T4 (Matthews, C. K., Kutter, E. M., Mosig, G.,and Berget, P.B., eds) pp. 277290, American Society for Microbiology, Wash., D. C. Hughes, M.B., Yee, A.M. F., Dawson, M., and Karam, J. D. (1987) Genetics 115,393-403 Lin, T.-C., Rush, J., Spicer, E. K., and Konigsberg, W. H. (1987) Proc. Natl. Acad. Sei. U. S. A. 84, 7000-7004 Sober, H. A. (1970) Handbook of Biochemistry: Selected Valu~s for Molecular Bwhgy, 2nd Ed., pp. C3-C25, Chemical Rubber Co., Cleveland
