Alkaline deoxyribonuclease induced by herpes simplex virus type 1: composition and properties of the purified enzyme

VIROLOGY

103,

Alkaline

493-501

(1980)

Deoxyribonuclease Induced Composition and Properties

by Herpes Simplex Virus Type 1: of the Purified Enzyme

MARJORIE STROBEL-FIDLER’

AND

Accepted February

BERTOLD FRANCKE’

27, 1980

The alkaline DNase inclueed by herpes simplex virus type 1 was purified 520-fold, ineluding ehromato~aph~ in the presence of high salt conce~ltrations. The purest ~trep~ration exhibits three comp0nent.s after electrophoresis under denaturing eonditions: a doublet of 90,000 daltons, a polgpeptide of 85,000 daltons-most likely a breakdown product-and small amounts of a 70,000~dalton protein. [%]Methionine added to infected cells labels exclusively the 90,000-dalton component, suggesting this as the molecular weight of the denatured nuclease. During sedimentation in 50 m&f salt the purified enzyme has an apparent molecular weight of ~~,OOO,as compared to globular marker proteins, indicating that the native protein is rod shaped. The response of the enzyme to mono- and divalent ions is similar to that reported by others (Hoffman and Cheng. 1977).The effect of pH appears to be rather on the enzyme than on the conformation of the substrate. The purest fraction exhibits endonuclease activity for supercoiled DNA under standard conditions (lo-fold less than esonuelease) and appears to degrade the ent?onucleol~ticall~ cleaved substrate processively. INTRODUCTION

The DNase activity induced in cultured cells after herpes simplex virus (HSV) infection (Keir and Gold, 1962) appears to be a DNA-specific nuclease with a high pH optimunl pro~lucing 5’-deo~yribonueleoside monophosphates {Morrison and Keir, 1968; Hoffman and Cheng, 1977). For HSV type 2 one temperature-sensitive mutant induces a DNase which is more heat labile than the wild-type enzyme (Francke et al., 1978), yet studies on revertants of the mutant have shown that the lesion is nonlethal (Moss et al., 1979). No similar mutant for HSV type 1 is available at this time. The enzyme of both types, as purified by Hoffman ancl Cheng (1979), has an associated endonuclease activity. No information is as yet available on the denatured molecular weight of the enzyme or its subunits. In t,his communication we report on the purifi’ Current address: Department of Biology, University of California, San Diego, La Jolla, Calif. 92037. 1 Author to whom reprint requests should be tKith-l2SS~d.

cation, composition, and properties of the nuclease induced by HSV type 1. MATERIALS

AND METHODS

Cebls, z%%s, arzd ~~?f~ct~~7~ c~~~~~t~o?~s. The HSV-1 (Glasgow strain 17) ts+ syni was grown and titered on BHK 21 ((~13) cells as described (Francke, 1977a, b). For the preparation of the enzyme extract BHK cells were infected at a multiplicity of 10 plaque-forming units per cell for 16 hr at 37”. E?Lxy)rre assays. Alkaline nuclease was assayed as described (Francke et al., 1978). Escherichia coli DNA polymerase I was assayed as described by Richardson et al. (1964). Protein was determined by the method of Lowry et a.1. (1951). P~~~ca~~o~~ procedure. A total of 1.4 x10” infected cells were scraped into the culture medium, washed once in Trisbuffered isotonic saline, and resuspended in 200 ml of buffer A (10 mW Tris-HCl, pH 7.5, 1 mil/l MgCX, 0.5 m&Z dithiothreitol). After addition of KC1 to 80 m&f

494

STROBEL-FIDLER

and NP40 to O.Z%, the lysate was homogenized with 20 strokes in a tight-fitting dounce homogenizer. Nuclei and cell debris were pelleted at 6000 g for 30 min and the pellet was reextracted with an equal amount of the same buffer. The combined supernatant fractions represent the crude extract. Soluble extract (Fraction I) was the supernatant after centrifugation at 100,000 g for 1 hr. The pellet obtained from an (NH&SO, cut between 25 and 55% saturation was resuspended in 100 ml of buffer A, dialyzed against three changes of buffer A (Fraction II), and applied to a 2.5 x lo-cm column of DEAE Sepharose in buffer B (same as buffer A, with 10% glycerol, 0.2% NP40, 20 mM potassium phosphate buffer, pH 7.5). During elution with a gradient from 20 to 300 mM potassium phosphate (400 ml total volume), the enzyme eluted between 100 and 200 mib’ (Fraction III). Phosphocellulose chromatography was performed as described by Weissbach et al. (1973), and the enzyme eluted as a sharp peak at 210 mM potassium phosphate (Fraction IV). Single-stranded DNA coupled to activated Sepharose was prepared as described by Poonian et al. (1971). Chromatography was performed in buffer C (same as buffer B except that TABLE

1

PURIFICATION0F ALKALINE NUCLEASE FROM HSV-~-INFECTED

Fraction

I II III IV V VI VII VIII

BHK CELLS

Stage Crude extract Soluble extract (NH,),SO, DEAE Sepharose Phosphocellulose DNA Sepharose Hydroxylapatite Glycerol gradient Norleucine Sepharose

Recovery (%I

Specific activity (unitsipg protein)”

100 99.1 113.6 109.8 50.1 29.4 17.6 13.6 6.8

0.010 0.021 0.045 0.187 3.100 3.750 5.200 5.200

Note. The purification of alkaline nuclease from 1.4 x lOi HSV-l-infected BHK cells was as described in detail under Materials and Methods. 0 One unit = degradation of 1 pg DNA/min at 37”.

AND FRAN’CKE

MgCl, and NP40 were omitted and 2 mM EDTA was present). The enzyme eluted at 170 mM potassium phosphate (Fraction V). After concentration by hydroxylapatite chromatography (Richardson et al., 1964) (Fraction VI) the enzyme was sedimented through linear 15 to 45% glycerol gradients (in 10 mM potassium phosphate, pH 7.5,0.5 mM dithiothreitol, compare Fig. 3). Centrifugation was in a Beckman SW 41 rotor for 72 hr at 37,000 rpm and 2”. Active fractions (Fraction VII) were pooled and applied to a norleucine-Sepharose column (Morris et al., 1979) in the presence of 1.5 M potassium phosphate (pH 7.5), 2 M NaCl, 10% glycerol, 0.5 mM dithiothreitol, and 1 ti EDTA. The enzyme was eluted in the same buffer with a decreasing potassium phosphate gradient with 2 M NaCl present throughout. This final preparation (Fraction VIII) was dialyzed against buffer B and stored in aliquots at -90”. A summary of the purification is shown in Table 1. Labeling with [“5S]methionine. Each 1.6 x lox cells was labeled either for 24 hr prior to infection or from 2 to 16 h after infection with [3”S]methionine (20 PCi [“5S]methionine/ml of Dulbecco modified Eagle’s medium with a fivefold reduced methionine content, containing 2% calf serum). The cells that had been labeled prior to infection received regular medium with 10% calf serum after infection. Purification of nuclease was as described above, except that the hydroxylapatite and norleucine-Sepharose steps were omitted, and the sizes of columns and the volumes of buffers used were reduced fivefold. Gel electrophoresis

and protein

markers.

Polyacrylamide gel electrophoresis of SDSdenatured proteins was as described by Heine et al. (1973), except that slab gels of 15% acrylamide crosslinked with 0.1% bisacrylamide were used. Gels were stained with Coomassie brilliant blue and photographed. For gels containing 35S-labeled proteins, autoradiography with X-ray film was performed after drying under vacuum. Molecular weight standards for denaturing gels were: myosin, /3-galactosidase, phosphorylase b, bovine serum albumin, and ovalbumin (obtained as a mixture from BioRad Laboratories). Marker proteins during

HSV-1 ALKALINE

glycerol gradient sedimentation wereE. coli DNA polymerase I (109,000 daltons, kindly provided by Dr. Inder Verma) and hemoglobin and cytochrome c (64,000 and 13,000 daltons, respectively, kindly provided by Dr. Tony Hunter). The first was identified by activity, the latter two by optical density. Po~yoma virus DIVA and alkal~~~~ sucrose gradient centrifitgation,. Unlabeled and 3”P-labeled polyoma virus form I DNA was prepared by Hirt extraction of infected mouse 3T3 cells followed by ethidium bromide-CsC1 equilibrium gradient centrifugation (Hunter and Francke, 1974). Alkaline sucrose gradient sedimentation was as described in the same reference, using a Beckman SW 56 rotor for 2 hr at 50,000 rpm and 5”. The form I DNA used for the experiments described consisted >95% super-coiled DNA as determined by a second equilibrium eentrifugation in an ethidium bromide-CsCl gradient, but contained 25% alkali labile material as judged after alkaline sucrose gradient sedimentation. Materials. DEAE Sephadex (A-25) and eyanogen bromide-activated Sepharose were from Pharmaeia, phosphoeellulose (P-11) from Whatman, hydroxylapatite and the reagents for denaturing polyacrylamide electrophoresis were from Bio-Rad Laboratories. RESULTS

Pwijication, Enzyme

Composition

and Size of the

495

DNase

- top

y9OK -85K -i’OK

---(40 K)

--BP3 FIG. 1. SDS-polyacrylamide gel electrophoresis of HSV nuclease fraction VIII: 30 ~1 Fraction VIII. containing ca. 10 pg of total protein, was denatured in P-mereaptoethanol and SDS, electrophoresecl on a IFi%, polyaerylamitle slab gel. and stained with Coomassie blue as described under Materials and Methods. Five molecular weight standards from 120,000 to 40,000 daltons were electrophoresed in a parallel lane. The numbers indicate the apparent molecular weights Y 10 :I for each component as determined by comparison with the standards. 40K refers to the position of a pofypeptide visable when larger amounts of protein were analyzed. BPB refers to the position of the bromophenol blue front.

The large-scale purification of the alkaline nuclease from HSV-1 infected BHK cells is summarized in Table 1, and a photograph of the final preparation (Fraction VIII) after electrophoresis in a denaturing polyacrylamide gel and Coomassie blue staining column in the presence of 2 M NaCl) it is shown in Fig. 1. Despite a greater than seems not to be necessary for enzymatic 500-fold purification over the crude extract, activity. Of the other three compone~lts~the three main components are displayed in the origin of and possible requirement for the gel. When larger amounts of Fraction VIII 70,000-dalton polypeptide remains unclear were loaded on the gel, a further com- (see below). The 85,000-dalton polypeptide ponent (40,000 daltons) was detectable. is most likely a breakdown product of the This latter polypeptide copurified with the YO,OOO-dalton polypeptide, since its relative nuclease through Fraction VII (compare amount increased during the final stages of also Fig. 3). But since it was removed the puri~cation at the expense of the during the final step (norleucine-Sepharose latter (not shown). At this stage of the

STROBEL-FIDLER

4%

A

B

CDEFGHI

AND FRANCKE J

FIG. 2. SDS-polyacrylamide gel electrophoresis of [‘“Slmethionine-labcletl proteins during the purification of alkaline nuclease from infectetl BHK cells: labeling ant1 purification as describetl in Table 2. Eyual amounts of nuclease activity acre electrophoreaetl in parallel lanes of a 1.5% polyacrylamide gel, as describetl in Fig. 1. Lanes A through E are &rived from preinfection label, ant1 F through J from postinfection label. A and F’ represent Fraction I. B ant1 G Fraction III. C: ant1 H Fraction IV, D ant1 I Fraction V, ant1 E ant1 J Fraction VII of the purification as tletailetl in Table 1. The numbers indicate the molecular weights x 10 ” for the protein standartls electrophoreaetl in parallel lanes. The result of a Z-week autoratliogrdphy exposure is shown.

purification the 90,000-dalton component consistently electrophoresed as a doublet for reasons not understood. To obtain further information as to the origin of the three stainable polypeptides in Fraction VIII, a small-scale purification of the TABLE

2

SPECIFIC RADIOACTIVITY (:‘iS cpm x lo-WNIT OF ENZYME) DURING THE PURIFICATION OF ALKALINE XWLEASE LABELED WITH [%]METHIONINE

Fraction”

Before infection

After

infection

Nofe. BHK cells lvere labeled with [:‘;‘Slmethionine for 24 hr prior to or from 2 to 16 hr after infection with HSV-1. The details for labeling and purification of proteins were as described untler Materials and Methotls. ” The fractions correspond to the purification steps tletailetl in Table 1.

nuclease was carried out after labeling of the protein with [:‘“S]methionine either before or after infection. The specific radioactivity of the preparations at selected purification stages is listed in Table 2, and autoradiographs of the corresponding fractions after denaturing gel electrophoresis are shown in Fig. 2. It is apparent that as of Fraction V (DNA Sepharose) practically all host polypeptides labeled before infection have been removed, and that the 90,000dalton polypeptide labeled after infection appears to be radiochemically pure as of Fraction VII (glycerol gradient). Since in neither case did a 70,000-dalton component appear, it cannot be decided whether the stainable band of this molecular weight represents a methionine-free protein of either cellular or viral origin, or whether this component copurified in the large scale preparation due to the higher total protein concentration. Compared to the 90,000 and 85,000 components, it appears to be present in submolar amounts, and we cannot exclude that it represents a serum protein

HSV-1 ALKALINE

5

IO

497

DNase

I5

20

FRACTION NUMBER FIG. 3. Glycerol gradient sedimentation analysis of alkaline nuclease: Fraction VI of the purification described in Table 1 was mixed with DNA polymerase I (Pol I), hemoglobin (Hb), and cytochrome c (Cyt c) and sedimented through a 15-45% glycerol gradient as described under Materials and Methods. In a parallel gradient Fraction VI nuclease was sedimented without markers. Of this gradient the fractions corresponding to the nuclease were analyzed by SDS-polyacrylamide gel electrophoresis as described in Fig. 1. A photograph of the Coomassie blue-stained gel is placed over the corresponding enzyme fractions in the figure.

which binds tightly to the nuclease. Patterns identical to the one shown in Fig. 1 were obtained from three independent largescale enzyme preparations, the only difference being variable amounts of the 85,000-dalton component. Taking all the evidence obtained so far together, we would like to suggest that the 90,000-dalton polypeptide represents the virus induced alkaline nuclease, which may or may not require an additional 70,000-dalton polypeptide for activity. The sedimentation rate of the native enzyme (Fig. 3) on the other hand suggests a molecular weight of 40,000 daltons compared to three globular marker proteins. Since the polypeptides present in Fraction VIII argue against a molecular weight of less than 70,000, it has to be concluded that the enzme is rod shaped rather than

globular in order to explain its sedimentation behavior. The sedimentation of the purest fraction (VIII not shown) was identical to that shown for fraction VI in Fig. 3, and is therefore not caused by some contaminant still present before norleucineSepharose chromatography. All following experiments were carried out with Fraction VIII. Properties

of the Puti$ed

Enzyme

The general properties of Fraction VIII were similar to those determined by Hoffmann and Cheng (19’77). Briefly, the optimum for sodium and potassium is between 20 and 30 mM, while higher concentrations inhibit (50% at 100 n&f) with the acetate salts being less inhibitory than the chloride salts. The activity is totally dependent on

498

STROBEL-FIDLER

AND FRANCKE

which may be compared to our Fraction V in purity, that it has an associated endonucleolytic activity, which copurified and cosedimented with the endonuclease at constant ratios. Our Fraction VIII exhibits also some endonuclease as demonstrated in Fig. 6. When present in equimolar amounts of DNA nucleotide, covalently closed, supercoiled polyoma virus form I DNA (3aPlabel) is degraded to acid-soluble material, almM CaC12 mM EGTA though at a rate IO-fold slower than linear DNA from BHK cells (3H label). ‘j2PFIG. 4. Inhibition of the alkaline n&ease by Ca’* Labeled linear DNA is indistinguishable and its reversal by EGTA: Standard nuclease reactions from “H-labeled linear DNA, while nicked were carried out with either 2 nu’l4 MgCl, (0) or 0.15 circular polyoma DNA form II (1.3 average mM MnCl, (X). In panel a the remaining activity after nicks per molecule) behaves intermediately the addition of the indicated amounts of CaCl, is plotted. In panel b a reaction containing 5 n&! MgCl, but is degraded more than half as efficiently and 2 mJ4 CaC1, (0) is titrated with the indicated as linear DNA. In order to determine amounts of EGTA and the recovery of n&ease activity whether nicking as an initial stage of is plotted. Activities are presented as percentage endonuclease action could be demonstrated, of the reaction containing 2 mM MgCl,, only. the products were analyzed by alkaline sucrose gradient sedimentation. As shown in Fig. 7, the remaining acid-precipitable the presence of sulfhydryl reagents and linear DNA decreases in single-strand size, divalent cations. Magnesium is the preferred ion with a broad optimum between 2 compatible with a distributive mode of and 12 mM, manganese can substitute action of the enzyme on this substrate as partially (60% of the activity with Mg2+ at described by Hoffmann and Cheng (1977) 0.15 mM Mn*+, with total inhibition above under standard reaction conditions with 0.5 mM). Calcium on the other hand is in- magnesium present as the only divalent hibitory even in the presence of magnesium cation. The polyoma form I DNA (rapidly or manganese as shown in Fig. 4a. This is specifically reversible by the Ca2+ chelator EGTA (Fig. 4b). The high pH optimum of the DNase (Morrison and Gold, 1968) may be a property of the enzyme itself or reflect its preference for single-stranded or partially denatured DNA as substrate. We therefore tested the effect of pH on the degradation of native and denatured DNA, with both Mg2+ and Mn2+ present (Fig. 5). Using single-stranded DNA as substrate the pH optimum for the nuclease was not lowered with respect to double-stranded PH DNA, in either case. This suggests that the FIG. 5. Influence of pH on the nuclease with singlehigh pH is required for the conformation of or double-stranded DNA as template: Standard nuclease the enzyme itself in order to be an efficient reactions were carried out with either 2 mW MgCl, nuclease, and raises the question whether (a) or 0.1 mfl4 MnCI, (b), except that 20 m&’ HEPES this is its physiological role. buffer was used instead of Tris-HCI. The HEPES Endonucleolytic

Activity

Hoffmann and Cheng (1979) have reported for their nuclease preparation,

had been was then potassium substrate by boiling

adjusted to the pH indicated with KOH and brought to 1 mW K-/m01 of HEPES with acetate (adjusted to pH ‘71. The [“HIDNA was used either native (0) or denatured for 5 min followed by quenching in ice (0).

HSV-1 ALKALINE

3H-BHK

499

DNaae

0 32P-BHK

DNA

DNA

\ \

8

0

\

\

\

0

0

\ \

0 024 NUCLEASE

(~1)

FIG. 6. Degradation of linear DNA and covalentlg closed circular DSA by the alkaline nucleaw: Standard wactions were carr%tl out for 15 min with the indicated amounts of nuclease Fraction VIII present. ‘LH-Labeled linear RHK DNA \vas either digested alone at 1% &ml. or at half that concentration ivith an equal amount of ‘“P-labeled covalrntly closed circular polyoma virus DSA folm I, nicked circular DNA II, or linear RHK DNA present. The percentage of total input ratlioactivit), acid Ixecipitable after incubation. is plotted.

sedimenting, 55 S) used in the experiment contained about 25% alkali-labile material sedimenting as single strands (slower peak, 16-18 S) (Fig. 7A). It is not due to nicks that may have occurred during storage, since reanalysis in ethidium bromide-CsCl equilibrium gradients showed that the preparation contained >95R supercoiled DNA. As the reaction proceeds (Figs. 7B and C), the ratio of 55 to 16-18 S material remains constant, and no gradual shift to smaller sizes is observed even for the single-stranded peak. Because of the failure to detect a kinetically stable nicked intermediate from supercoiled DNA or a gradual decrease in single-stranded size, it has to be concluded that this substrate, once attacked by the endonucleolytic activity, is degraded rapidly and processively by, the exonucleolytic activity. DISC’USSION

The analysis of a greater than 500-fold purified preparation of the HSV-l-induced nuclease presented here has shown that

several polypeptides copurify with the enzymatic activity. For instance a 40,000dalton protein appears to stick tightly to the enzyme during large-scale purification and is removed only by chromatography in the presence of high salt concentrations (norleutine-Sepharose column) without loss of enzymatic activity. Cochromatography of endonuclease and exonuclease activities, for instance during separation procedures involving lower salt concentrations, do not prove that the activities are carried by a single polypeptide. Furthermore, radiochemical purity after purification of [%Imethionine-labeled proteins can be deceiving, since in this case it did not reveal the presence of the 70,000-dalton polypeptide. The enzyme itself appears to bind frequently and strongly to other proteins. The viral DNA polymerase, for instance, does not separate completely from the nuclease until Fraction IV (phosphocellulose). Another example is bovine serum albumin, which could not be used as a sedimentation marker, because the nuclease bound to it and shifted in position. The relatively high

500

STROBEL-FIDLER

AND FRANCKE

In HSV type 2, a temperature-sensitive mutant for the nuclease exists (Francke et al., 19781,and studies with revertants of this mutant (Moss et al., 1979) have shown that, at least in HSV-2, the nut mutation appears to be nonlethal. For HSV-1, no similar mutation has been defined yet, and it is unknown whether the enzyme is essential for viral replication. It has been suggested (Hoffmann and Cheng, 1977) that the enzyme may degrade cellular DNA in order to provide deoxynucleotides for viral DNA synthesis. There is no published evidence for the de~adation of cell DNA during HSV infection, and those nucleolytic activities observed during cell-free viral DNA synthesis (Franeke, 19?7a, b) degrade the viral DNA exclusively and not the cellular DNA. We would like to suggest therefore that the endo- and exonucleolytic properties of the enzyme might be reactions which the enzyme can perform 1:n vitro, especially at high pH, but that its in viva function FRACTION NUMBER might be different. It is premature to specuFIG. ‘7. Alkaline sucrose gradient sedimentation of late on the nature of the possible in vivo reaction products derived from linear and covalently function of the nuclease, but it shares some closed circular DNA: A m&ease reaction was carried properties with other known enzymes. The out as described in legend to Fig. 6 with 2 ~1 enzyme inhibition by calcium ions is similar to the $H-BHK DNA and 32P-polyoma form I DNA present. behavior of the ret BC nuclease from At 0, 60, and 90 min the reaction was terminated Escherichia. co&i (Rosamond et ab., 1978), by the addition of 10 m&4 EDTA. After extraction and its conformation resulting in a lower S with phenol, the reaction products were analyzed by alkaline sucrose gradient sedimentation as deseribed value than expected from its molecular under Materials and Methods. After collection of the weight is reminiscent of DNA helicases gradients, the acid-precipitable radioactivity was (Kohn et al., 1978). We have attempted to determined. Sedimentation was from right to left. demonstrate other enzymatic activities in the purified nuclease preparation. We were unable to detect DNA polymerase or protein concentrations throughout the large- nucleotide triphosphatase (both for deoxyscale preparation may have contributed to and ribonucleotides, with or without the the amount of protein-protein binding. As addition of unspecific or viral DNA) under a explained in detail under Results, of the variety of conditions. These being negative three Coomassie blue-staining components results, it is still possible that for its in Fraction VIII after denaturing gel physiological function the nuelease has to electrophoresis (Fig. 1) the doublet at interact with other proteins in infected 90,000 daltons most likely represents the cells which have been removed during viral alkaline nuclease, while the 85,000- purification. dalton component appears to be derived ACKNOWLEDGMENTS from it by proteolytic degradation, We have to leave the question open as to whether the The authors would like to thank Drs. B. Sefton and 70,000-dalton polypeptide of unknown origin T. Taggart for advice on gel electrophoresis and is required for enzymatic activity, or B. Garrett for excellent technical assistance. This whether it represents a strongly binding work was supported by Grant AI 15644, awarded by the National Institutes of Health. contaminant.

HSV-1 ALKALINE REFERENCES FRANCKE, B. (1977a). Cell-free synthesis of herpes simplex virus DNA: Conditions for optimal synthesis. Biochemistry 16, 5655-5664. FRANCKE, B. (1977b). Cell-free synthesis of herpes simplex virus DNA: Structure of the in vitro product and nucleolytic degradation. Biochemistry 16, 5664-5670. FRANCKE, B., Moss, H., TIMBURY, M. C., and HAY, J. (1978). Alkaline DNase activity in cells infected with a temperature-sensitive mutant of herpes simplex virus type 2. J. Viral. 26, 209-213. HEINE, J. W., HONESS, R. W., CASSAI, E., and ROIZMAN, B. (1973). Proteins specified by herpes simplex virus. XII. The virion polypeptides of type 1 strains. J. Viral. 14, 640-651. HOFFMANN, P. J., and CHENG, Y. C. (1977). The deoxyribonuclease induced after infection of KB cells by herpes simplex virus type 1 or type 2. I. Purification and characterization of the enzyme. J. Biol. Chem. 253, 3557-3562.

HOFFMAN, P. J., and CHENG, Y. C. (1979). DNase induced after infection of KB cells by herpes simplex virus type 1 or type 2. II. Characterization of an associated endonuclease activity. J. Viral. 32, 449-457. HUNTER, T., and FRANCKE, B. (1974). In vitro polyoma DNA synthesis: Characterization of a systern from infected 3T3 cells. J. Viral. 13, 125-139. KEIR, H. M., and GOLD, E. (1962). Deoxyribonucleic acid nucleotidyl-transferase and deoxyribonuclease from cultured cells infected with herpes simplex virus. Biochim. Biophys. Acta 72, 263-276. KUHN, B. ABDEL-MONEM, M., and HOFFMANNBERLING, H. (1978). DNA helicases. Cold Spring Harbor LOWRY,

Symp. Quant. Biol. 43, 63-67. D. H.,

ROSEBROUGH,

N. J.,

FARR,

A.

L.,

DNase

501

and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-271. MORRIS, F. M., HAMA-INABA, H., MACE, D., SINHA, N. K., and ALBERTS, B. (1979). Purification of gene 43, 44, 45, and 62 proteins of the bacteriophage T4 replication apparatus. J. Rio/. Chem. 14, 6787-6796. MORRISON, J. M., and KEIR, H. M. (1968). A new DNA-exonuclease in cell infected with herpes virus: Partial purification and properties of the enzyme. J. Gen. Viral. 3, 337-347. Moss, H., CHARTRAND, P., TIMBURY, M. C., and HAY, J. (1979). Mutant of herpes simplex virus type 2 with temperature-sensitive lesions affecting virion thermostability and DNase activity: Identification of the lethal mutation and physical mapping of the nut- lesion. J. Viral. 32, 140-146. POONIAN,S. M., SCHLABACH,A. J., and WEISSBACH, A. (1971). Covalent attachment of nucleic acids to agarose for affinity chromatography. Biochemistl-y 10, 424-427. RICHARDSON,C. C., SCHILDKRAUT,C. L., APHOSIAN, H. V., and KORNBERG, A. (1964). Enzymatic synthesis of deoxyribonucleic acid XIV. Further purification and properties of deoxyribonucleic acid polymerase of Escherichia coli. J. Biol. Chem. 239, 222-231. ROSAMOND,J., ENDLICH, B., TELANDER, K. M. and LINN, S. (1978). Mechanism of action of the type-l restriction endonuclease, Eco B, and the recBC DNase from Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 43, 1049-1058. WEISSBACH, A., HONG, S., AUCKER, J., and MULLER, R. (1973). Characterization of herpes simplex virus induced deoxyribonucleic acid polymerase. J. Biol. Chem. 248, 6270-6277.