THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 262, No. 9, Issue of March 25, pp. 4260-4266, 1987 0 1987 by The American Society of Biological Chemists, Inc Printed in U.S. A. Interaction between theDNA Polymerase and Single-stranded DNA- binding Protein (Infected Cell Protein 8) of Herpes Simplex Virus l* (Received for publication, September 22, 1986) Michael E. O’DonnellS, Per EliasQ,Barbara E. Funnellll, and I. R. Lehman From the Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305 The herpes virus-encoded DNA replication protein, ICP8 completely inhibits replication of singly primed ssDNA infected cell protein 8 (ICPS), binds specifically to templates by the herpespolymerase. On the other hand, ICP8 single-stranded DNA with a stoichiometry of one ICPS strongly stimulates replication of duplex DNA. It does so, molecule/l2 nucleotides. In the absence of single- however, onlyin the presence of anuclear extract from stranded DNA, it assembles into long filamentous herpes-infected cells. structures. Binding of ICPS inhibits DNA synthesis by the herpes-induced DNA polymerase on singly primed EXPERIMENTALPROCEDURES single-stranded DNA circles. In contrast, ICPS greatly Materials-DNase I and venom phosphodiesterase were obtained stimulates replication of circular duplex DNA by the from Worthington. The syntheticoligodeoxynucleotide (44-mer) was polymerase. Stimulation occurs only in the presence of synthesized by the solid-phase coupling of protected phosphoramidate a nuclear extract from herpes-infected cells. Appear- nucleoside derivatives (11).3H-Labeled #X ssDNA was a gift from ance of the stimulatory activity in nuclear extracts Dr. R. Bryant (this department). pMOB45 (10.5 kilobases) linear coincides closely with the time of appearance of her- duplex plasmid DNA was a gift from Dr. D. Julin (this department). pes-induced DNA replication proteins including ICPS M13mp18 RF I DNA and Ml3rnplSoris RF I DNA were prepared as and DNA polymerase. A viral factor(s) may therefore described (12); HSV-1 DNA polymerase was prepared as described be required to mediate ICPS function in DNA replica- (10). Heparin-Sepharose was purchased from Pharmacia P-L Bio- tion. chemicals. Glutaraldehyde (5076, v/v) and Alcian blue 86s were obtained from Electron Microscope Sciences. Bio-Gel A-5m was obtained from Bio-Rad. All other chemicals, nucleic acids, and en- zymes not described here were from sources or prepared as in the preceding paper (10). The herpes simplex virus 1 (HSV-1)’-encoded ICP8 is a Buffers-Buffer A was 20 mM Tris-Cl (pH 7.5), 6 mM MgC12, 4% 128-kilodalton DNA-binding protein (1, 2) that is essential (w/v) glycerol, 0.1 mM EDTA, 40 pg/ml bovine serum albumin, 5mM for viral DNA replication (3-5). Conditional lethal mutants DTT. Buffer B was 20 mM Hepes/Na+ (pH 7.6), 0.5 mM DTT, 0.5 in the ICP8 gene have a quick-stop phenotype, indicating a mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 10% (w/v) glyc- direct role of ICP8 in replicationfork movement (3). ICP8 is erol. Buffer c was 50 mM Tris-C1 (pH 7.5), 0.5 mM DTT, 0.1 mM (6), EDTA, 150 mM (NH,),SO,. Buffer D was 25 mM Tris-C1 (pH 7.2), abundant in nuclei the site of HSV-1 replication (7); and 10 mM MgCl,, 5% glycerol. Buffer E was 25 mM Tris-C1 (pH 7.5), 1 purified ICP8 binds tightly and cooperatively to ssDNA (8). mM DTT, 0.5 mM EDTA, 10% (w/v) glycerol. Buffer F was 20 mM Although the exact role of ICP8 in viral replication is still Tris-C1 (pH 7.5), 20 mM NaCl, 8 mM MgCl,, 1 mM DTT. unknown, its cellular abundance and binding to ssDNAsug- Purification of ICP8-1CP8 was purified by a modification of the gest that ICP8 is functionally analogous to the Escherichia method of Powell et al. (13). HSV-1-infected Vero cells (50 g, wet coli SSB (9). E. coli SSB binds tightly and cooperatively to weight) from 100 roller bottles were harvested 18 h after infection ssDNA, creating an activatedsurface for replication by DNA with HSV-1 as described (12). Nuclei were prepared and extracted with 1.7 M NaCl, and the extractwas fractionated by phosphocellulose polymerase I11 holoenzyme (9). Similarly, SSB strongly stim- chromatography as described (12). At all stages of purification, col- ulates theHSV-1-encoded DNA polymeraseon singly primed umn fractions were analyzed by 7.5% SDS-polyacrylamide gel elec- ssDNA templates(10). In experimentsdescribed in this paper, trophoresis followed by staining with Coomassie Blue; ICP8-contain- we have examined theeffect of ICP8 on DNA replication by ing fractions were identified from the known mobility of ICP8 (14). the herpes-inducedDNA polymerase. In contrast to SSB, ICP8 was eluted from the phosphocellulose column at 0.15 M NaCl ~~ as previously reported (12, 13) and was approximately 50% pure at * This research was supported by Grant GM06196 from the Na- this stage. The pooled phosphocellulose fractions were dialyzed tional Institutes of Health. The costs of publication of this article against buffer B and applied to a ssDNA-cellulose column (20-ml bed were defrayed in part by the payment of page charges. This article volume) equilibrated with buffer B. The ssDNA-cellulose column was must therefore be hereby marked “aduertisement” in accordance with eluted with a linear gradientfrom 0.1 to 1.0 M NaCl in a total volume 18 U.S.C. Section 1734 solely to indicate this fact. of 240 ml. Fractions (2 ml) were collected. The ICP8eluted in a broad $ Fellow of the Helen Hay Whitney Foundation. Present address: peak. Fractions containing ICP8 were pooled, dialyzed against buffer Dept. of Microbiology, Cornel1 University Medical School, 1300 York C containing 5% (w/v) glycerol, and concentrated to 4 mg/ml in 0.5 Ave., New York, NY 10021. ml by ultrafiltration in a Centricon 30 (Amicon). At this stage, ICP8 4 SuDDorted by a fellowship from the Knut and Alice Wallenberg was over 98% pure but was contaminated with a potent DNA exo- Foundation, Sweden. nuclease (assayed by digestion of 5.4-kilobase linear duplex DNA). 1 SuDDorted bv a fellowshiD from the Medical Research Council The exonuclease contaminant was completely removed by sedimen- of Canida. Present address: Lab. of Biochemistry, NCI, NIH, Be- tation of ICP8 in a glycerol gradient. The ICP8 preparation was thesda, MD 20892. layered onto two 10.6-ml 10-30% glycerol gradients in buffer C and The abbreviations used are: HSV, herpes simplex virus; Hepes, centrifuged at 40,000 rpm for 40 h at 4 “C in a Beckman SW41.Ti 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid @X, bacterio- rotor. After centifugation, 0.2-ml fractions were collected from the phage 4x174; ssDNA, single-stranded DNA; RF I, closed circular bottom of the tubes. Total protein was measured by a modification duplex DNA; RF 11, circular duplex DNA with a nick in one strand of the method of Bradford (see Ref. 15). A total of 800 pg of ICP8 SDS, sodium dodecyl sulfate; DTT, dithiothreitol; SSB, E. coli single- was obtained by this procedure. stranded DNA-binding protein; ICP8, infected cell protein 8. Because of the relatively poor recovery of ICP8 following ssDNA- 4260 HSV-1 Single-stranded DNA-binding Protein 4261 cellulose chromatography and glycerol gradient sedimentation, we amount of singly primed @XssDNA that was fully replicated in 30 have devised an alternativeprocedure using heparin-Sepharose chro- min at 30 "C by a given amount of enzyme as described in the matography. The peak phosphocellulose fractions were pooled and preceding paper (10). precipitated by addition of (NH,),SO, to 30% saturation (at 0°C). 3'4' Exonuclease Assay-The 3'+5' exonuclease activity of the This procedure resulted in removal of several polypeptide contami- herpes DNA polymerase was assayed using 3.1 pmol (as template nants toyield an ICP8 preparation thatwas over 80% pure.The pellet molecules) of 3"labeled hairpin templates having paired or unpaired (7 mg of protein) was dissolved in 0.75 ml of buffer D and dialyzed 3' termini in 12.5 p1 of buffer A containing 83 fmol of polymerase against 1 liter of buffer E. Four mg of the dialyzed ICP8 was diluted and 60 mM NaCl as described in the preceding paper (10). to 8 ml in buffer E and applied to a heparin-Sepharose column (3-ml Preparation of Nuclear Extract-Nuclei from lo9 18-hr infected or bed volume) equilibrated with buffer E. The column was eluted with mock-infected cells were prepared, resuspended in 10ml of buffer as a linear gradient from 0.1 to 0.6 M NaCl in a total volume of 40 ml of described (12), and stored as1-ml aliquots at -80 "C. A 1-ml sample buffer E. Fractions (0.35 ml) were collected and analyzed for ICP8 of nuclei was thawed slowly on ice and then lysed by addition of 85 and exonuclease activity. ICP8 was eluted from the column at 0.3 M p1 of 5 M NaCl followed by a 30-min incubation on ice. Insoluble NaCl as a sharp peak in 60% yield. The leading half of the ICP8peak chromatin was pelleted by centrifugation at 15,000 rpm in a Beckman was homogeneous (>99% pure) and was completely free of exonucle- JA-20 rotor for 30 min at 4 "C. The supernatant was dialyzed 3 h ase activity; however, the trailing half of the ICP8 peak was slightly against 1 liter of buffer Econtaining 25 mM NaCl at 4 "C. The contaminated with exonuclease. Exonuclease-free ICP8 prepared by dialyzed extract (600 pl) was concentrated to 3 mg/ml (200 p1 total) both procedures was used interchangeably in the experiments to be by ultrafiltration in a centrifuge (Centricon 10).