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Pseudorabies virus DNA-binding protein stimulates the exonuclease activity and regulates the processivity of pseudorabies virus DNase
BBRC Biochemical and Biophysical Research Communications 293 (2002) 1301–1308 www.academicpress.com
Pseudorabies virus DNA-binding protein stimulates the exonuclease activity and regulates the processivity of pseudorabies virus DNase Chien-Yun Hsiang* Department of Microbiology, China Medical College, 91 Hsueh-Shih Road, Taichung 404, Taiwan Received 16 April 2002
Abstract The pseudorabies virus (PRV) DNase is an alkaline exonuclease and endonuclease, which exhibits an Escherichia coli RecBCD-like catalytic function. The PRV DNA-binding protein (DBP) promotes the renaturation of complementary single strands of DNA, which is an essential function for recombinase. To investigate the functional and physical interactions between PRV DBP and DNase, these proteins were purified to homogeneity. PRV DBP stimulated the DNase activity, especially the exonuclease activity, in a dose-dependent fashion. Acetylation of DBP by acetic anhydride resulted in a loss of DNA-binding ability and a 60% inhibition of the DNase activity, suggesting that DNA-binding ability of PRV DBP was required for stimulating the DNase activity. PRV DNase behaved in a processive mode; however, it was converted into a distributive mode in the presence of DBP, implying that PRV DBP stimulated the dissociation of DNase from DNA substrates. The physical interaction between DBP and DNase was further analyzed by enzyme-linked immunosorbent assay, and a significant interaction was observed. Thus, these results suggested that PRV DBP interacted with PRV DNase and regulated the DNase activity in vitro. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Acetic anhydride; Chemical modification; DNase; DNA-binding protein; Endonuclease; Exonuclease; Physical interaction; Processivity; Pseudorabies virus; Recombination
Pseudorabies virus (PRV) is a member of Alphaherpesvirinae [1]. Alphaherpesviruses, such as herpes simplex virus type 1 (HSV-1), varicella-zoster virus, and PRV, have a closely similar gene arrangement and share a considerable amino acid sequence homology in their gene product [2]. During PRV DNA replication, seven proteins, including origin-binding protein, DNA polymerase/accessory complex, helicase/primase complex, and DNA-binding protein (DBP), are interacted and promote the rolling circle replication [3]. Additionally, a set of enzymes involved in DNA repair and nucleotide metabolism is expressed during viral replication. These enzymes are thymidine kinase, ribonucleotide reductase, deoxyuridine triphosphate nucleotidohydrolase, uracil glycosylase, and DNase [3].
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Fax: +886-4-2205-3764. E-mail address: [email protected] (C.-Y. Hsiang).
PRV DNase is a homolog of HSV-1 alkaline nuclease [4]. HSV-1 alkaline nuclease is not essential for DNA replication [5]. It is thought to provide nucleotides for HSV-1 DNA synthesis by degrading host cellular DNA [6]. It is also required for the efficient processing of viral DNA replication intermediates and for the egress of capsids from the nucleus [7,8]. Previous studies indicated that PRV DNase is able to cleave the double-stranded (ds) and single-stranded (ss) DNAs under the alkaline condition containing magnesium. Additionally, it exhibits an Escherichia coli RecBCD-like catalytic function with 50 -exonuclease, 30 -exonuclease, and endonuclease activities [9,10]. These and those results suggested that PRV DNase might play a role in the process of recombination and replication during PRV infection. PRV DBP is a homolog of HSV-1 infected cell polypeptide 8 (ICP8) [11]. ICP8 is essential for viral DNA synthesis [12]. It binds ssDNA rapidly and cooperatively [13]; it also promotes the renaturation of
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complementary single strands of DNA, which is an essential function of recombinase [14]. Several examples of physical and functional interactions between ICP8 and other enzymes have been described [15]. ICP8 interacts with the extreme C terminus of the origin-binding protein (UL9), greatly stimulating its DNA-dependent ATPase and DNA helicase activities [16]. Moreover, ICP8 stimulates the DNA polymerase/accessory complex [17] and assists the helicase/primase complex by preventing the reannealing of complementary single strands produced by its helicase action [18]. These results indicated that ICP8 might play an important role in the process of recombination and replication during herpesvirus infection. Since the HSV-1 ICP8 forms a complex with alkaline nuclease and colocalizes within the nuclear replication compartments [19,20], it is interesting to analyze the functional interaction between these two proteins in this study. Our data suggested that PRV DBP was associated with PRV DNase and this interaction might play an important role in viral recombination.
Materials and methods Proteins. Recombinant PRV DNase and DBP were expressed in E. coli BL21 (DE3) pLysS strain by transforming pET-DNase and pET/ DBP, respectively, to produce N-terminal fusions with six histidine residues. These proteins were purified to apparent homogeneity as described previously [9,11]. To ensure the correct folding of PRV DNase and DBP during purification, we carried out the DNase assay and nitrocellulose filter binding assay in each lot of proteins. The E. coli single-stranded DNA-binding protein (SSB) was purchased from United States Biological. DNase assay. The endonuclease and exonuclease activities of PRV DNase were assayed by incubating supercoiled and linear dsDNA, respectively, with DNase [9]. The 10-ll reaction mixtures, containing 0:5 lg of supercoiled pUC18 dsDNA or EcoRI-linearized pUC18 dsDNA, various amounts of PRV DNase, and DNase buffer (2 mM MgCl2 , 10 mM 2-mercaptoethanol, and 50 mM Tris/HCl, pH 9.0), were incubated at 37 °C for 5 min. The reactions were terminated by adding 1 ll of 10 stop buffer (50% glycerol, 1% SDS, 0.1% bromophenol blue, and 100 mM EDTA), analyzed by 1.2% agarose gels, and visualized by ethidium bromide. The amount of supercoiled or linear DNA banding on an agarose gel was quantitated by densitometric scanning. Lysine modification. The lysine was modified by acetic anhydride as described previously [21]. Purified DBP (0.1 ml) was mixed with an equal volume of saturated sodium acetate in DBP storage buffer (20% glycerol, 0.5 mM dithiothreitol, 0.2% NP-40, 10 lM ZnCl2 , 0.1 mM phenylmethylsulfonyl fluoride, and 50 mM Tris/HCl, pH 8.0). After the addition of 2 ll acetic anhydride, the mixture was incubated at 4°C for 30 min. Various amounts of acetic anhydride were then added and the mixture was incubated at 4 °C for another 30 min. Nitrocellulose filter binding assay. The formation of protein–DNA complexes was measured by using alkali-treated nitrocellulose filter as described previously [11]. Briefly, nitrocellulose filters (Hybond-C super; Amersham) were soaked in 0.5 M KOH for 20 min at room temperature, washed with distilled deionized water, and stored in 100 mM Tris/HCl (pH 7.6) at 4 °C. Neither ssDNA nor dsDNA was retained on the alkaline-treated filter, but protein–DNA complex was efficiently retained. The pUC18 dsDNA was labeled with [a-35 S]dATP by nick translation [22] and denatured to ssDNA by boiling for 5 min
and chilling on ice. The reaction mixture contained 100 ng of 35 S-labeled pUC18 ssDNA and various amounts of DBP in the DNAbinding buffer (1 mM EDTA, 150 mM NaCl, and 10 mM Tris/HCl, pH 7.5). A 100-ll sample of this mixture was incubated at 25 °C for 20 min and then applied to the alkali-treated nitrocellulose filters by using BioDot microfiltration units (Bio-Rad). The filters were washed twice with DNA-binding buffer and dried; the radioactivity retained on filters was determined with the scintillation counter (Beckman). Processivity of DNase. Processivity of DNase was performed as described previously [23]. PRV DNase (0.75 pmol) was mixed with 0:5 lg 35 S-labeled pUC18 dsDNA in 100 ll DNase buffer. After a 2-min incubation at 37 °C, a 50-fold excess of unlabeled pUC18 dsDNA was added and incubated at 37 °C as indicated. The undigested DNA was precipitated by adding 5 ll of 5 mg/ml bovine serum albumin (BSA) and 100 ll of 5% (w/v) trichloroacetic acid (TCA). The supernatant, containing acid-soluble nucleotides, was collected and the radioactivity of the nucleotides was determined with the scintillation counter (Beckman). Antibodies. The polyclonal antibodies (Abs) against DNase and DBP were prepared as described previously [24]. Briefly, Balb/c mice were injected with 0.5 ml pristane per mouse intraperitoneally. Two weeks later, 10 lg of protein was emulsified with an equal volume of Freund’s complete adjuvant (Gibco BRL) and injected subcutaneously. The protein boosted the mouse at a two-week interval twice. After the final immunization, 5 105 myeloma cells were injected intraperitoneally to each mouse, and the ascite, containing Abs, was collected daily for 5–8 days. Enzyme-linked immunosorbent assay (ELISA). The physical interaction between proteins was assayed by ELISA as described previously [25]. Microtiter plates (Costar) were incubated at 4 °C overnight with 50 ll of 10 ng=ll purified DBP or DNase, which was diluted in 0.05 M carbonate buffer (pH 9.6), as first layers. The wells were rinsed with 200 ll of washing buffer (0.5% Tween 20 in phosphate-buffered saline), and blocked with 200 ll blocking buffer (5% BSA in washing buffer) by incubating at 37 °C for 30 min. The absorbed protein in each well was challenged with various amounts of the second protein dissolved in 100 ll of blocking buffer and incubated at 37 °C for 1 h. The wells were then washed three times with washing buffer, and 50 ll of diluted mouse Ab, raised against the second protein, was added to each well and incubated at 37 °C for 1 h. After three washes with washing buffer, a 23,000-fold dilution of the goat anti-mouse IgG that was conjugated with peroxidase (Sigma) was added to each well and incubated at 37 °C for 1 h. Following three washes, 50 ll of the chromogenic substrate, 2; 20 -azinobis(3-ethylbenzthiazoline-sulfonic acid), was added to each well and incubated at 37 °C for 15 min. The absorbance was read at 405 nm in an ELISA plate reader.
Results and discussion Effect of PRV DBP on the PRV DNase activity Interactions between herpesviral DNase and other proteins have been reported in several studies [19,26]. To test whether the PRV DBP was able to influence the DNase activity, the exonuclease and endonuclease activities of PRV DNase were analyzed in the presence of DBP. The concentration of DNase used in this study was 0.5 pmol, which showed a limited DNase activity, and the DNase activity induced by DBP could be observed under this condition. Fig. 1 shows that the concentrations of DBP in excess of those of the PRV DNase (> 0:05 lM) stimulated the exonuclease activity of DNase up to 3.27-fold and this phenomenon displayed a dose-response manner. However, there was a slight
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Fig. 1. Effect of PRV DBP on the DNase activity. (A) Endonuclease and exonuclease activities of DNase induced by DBP. Lanes 1–5 represent the endonuclease activity by using supercoiled pUC18 dsDNA as the substrate. Lanes 6–10 represent the exonuclease activity by using linear pUC18 dsDNA as the substrate. Purified PRV DNase (0.5 pmol) was mixed with 0:5 lg dsDNA and 0 (lanes 2 and 7), 1 (lanes 3 and 8), 5 (lanes 4 and 9), or 10 (lanes 5 and 10) pmol of PRV DBP in the DNase buffer and incubated at 37 °C for 5 min. Lanes 1 and 6 contained no protein in the reaction. (B) Endonuclease and exonuclease activities of PRV DBP alone. Lanes 1–4 represent the endonuclease activity by using supercoiled pUC18 dsDNA as the substrate. Lanes 5–8 represent the exonuclease activity by using linear pUC18 dsDNA as the substrate. 0:5 lg dsDNA was mixed with 0 (lanes 1 and 5), 1 (lanes 2 and 6), 5 (lanes 3 and 7), or 10 (lanes 4 and 8) pmol of PRV DBP in the DNase buffer and incubated at 37 °C for 5 min. The resulting complex was analyzed by 1.2% agarose gels. Supercoiled and linear DNAs are indicated by arrows. (C) Quantitative analysis of DNase activity induced by PRV DBP. The amount of supercoiled or linear DNA was quantitated by Phoretix 1D program. Relative DNase activity was calculated by dividing the amount of DNase-treated DNA by the amount of DNase and DBP-treated DNA.
stimulation (1.48-fold) on the endonuclease activity in the presence of DBP. PRV DBP alone did not show the endonuclease and exonuclease activities (Fig. 1B). It showed a limited binding ability to dsDNA, which consisted of previous result [27]. To exclude the nonspecific stimulation of PRV DNase by another DNAbinding protein, we replaced the DBP with E. coli SSB and carried out the similar assay. No significant enhancement on both activities was observed (Fig. 2). Therefore, PRV DBP stimulated the exonuclease activity of PRV DNase, and this interaction was specific. To determine whether the stimulation of PRV DNase by DBP was due to an increase in the rate of DNA hydrolysis, time course experiments were carried out in the presence or absence of PRV DBP. The reaction mixtures, containing 0.5 pmol of PRV DNase, 0:5 lg linear DNA, and/or 10 pmol PRV DBP, were incubated
at 37 °C and stopped at indicated times. The concentration of DNase used in this study was 0.5 pmol, which showed a limited exonuclease activity, and the DNase activity induced by DBP could be observed under this condition. Fig. 3 shows that few DNAs were digested in the absence of DBP; however, the amount of digested DNA was increased in the presence of DBP as the incubation time was prolonged. DBP alone did not show the exonuclease activity. Therefore, PRV DBP displayed a stimulatory effect on the kinetics of DNA hydrolysis by PRV DNase. Effect of DNA-binding ability of PRV DBP on the DNase activity Basic and aromatic amino acid side chains have been shown to be involved in the interaction of prokaryotic
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Fig. 2. Effect of E. coli SSB on the PRV DNase activity. (A) Endonuclease activity. Purified PRV DNase (0.5 pmol) was mixed with 0.5 lg supercoiled pUC18 dsDNA and 0 (lane 1), 1 (lane 2), 5 (lane 3), or 10 (lane 4) pmol of E. coli SSB in DNase buffer and incubated at 37 °C for 5 min. (B) Exonuclease activity. Purified PRV DNase (0.5 pmol) was mixed with 0:5 lg linear pUC18 dsDNA and 0 (lane 1), 1 (lane 2), 5 (lane 3), or 10 (lane 4) pmol of E. coli SSB in DNase buffer and incubated at 37 °C for 5 min. The resulting complex was analyzed by 1.2% agarose gels. Supercoiled or linear DNA was indicated at the right. Lane C contained no protein in the reaction.
and eukaryotic single-stranded DNA-binding proteins with DNA [28–30]. To analyze whether the lysine residues were involved in DNA-binding ability of PRV DBP, and whether the DBP enhanced the DNase activity via binding to DNA, we carried out the chemical modification. The e-amino groups of lysine of PRV DBP were modified with acetic anhydrides. This reaction converts the positive charge of the amino groups to zero charge [31]. The DBP was treated with various amounts of acetic anhydride under alkaline conditions and the DNA-binding ability of DBP was evaluated by nitrocellulose filter binding assay. Fig. 4 shows that PRV DBP was acetylated by acetic anhydride in a dosedependent manner. Additionally, the acetylation of DBP by acetic anhydride resulted in a loss of DNAbinding ability, compared with the control sample. Thus, these results suggested that lysine residues were required for the interaction between PRV DBP and DNA. The DNase activity was further analyzed in the presence of wild-type DBP, acetic anhydride-modified
DBP, or acetic anhydride-modified E. coli SSB. Wildtype DBP enhanced the DNase activity up to 5-fold. In contrast, acetic anhydride-modified DBP exhibited a 60% inhibition on the DNase activity (Fig. 5). Acetic anhydride-modified E. coli SSB did not influence the DNase activity, which indicated that the inhibition of DNase activity by acetic anhydride-treated DBP was not caused by the residual acetic anhydride. Thus, these results suggested that DNA-binding ability of PRV DBP was required for stimulating the DNase activity. PRV DBP enhanced the PRV DNase activity. The lysine-modified DBP lost not only its DNA-binding ability, but also its ability to stimulate DNase activity, which suggested that DNA-binding ability of PRV DBP was important for stimulating the PRV DNase activity. Additionally, it also indicated that lysine residues might be involved in DNA-binding ability of PRV DBP and the functional interaction between PRV DBP and DNase. The N-terminally truncated Epstein–Barr virus (EBV) major DNA-binding protein (mDBP), which lost its DNA-binding ability, inhibits the DNase activity in vitro [23]. The exonuclease activity encoded by EBV DNase is also inhibited when this enzyme is associated in vivo with mDBP [26]. These studies suggested that the inhibition of DNase activity by mDBP might be a kind of viral self-protective mechanism to protect the viral DNA from viral DNase attack during the productive viral DNA synthesis. Bacteriophage T4 SSB strongly stimulates the exonuclease activity of T4 RNase H and converts it into a non-processive nuclease, suggesting that T4 SSB controls T4 RNase H degradation of RNA primers and adjacent DNA during each lagging strand cycle [32]. HSV-1 UL9 protein and ICP8 form a tight complex and this interaction greatly stimulates the rate and extent of DNA unwinding catalyzed by the UL9 protein [33]. This study showed the functional and physical interactions between PRV DBP and DNase. However, the biological role of functional interaction between PRV DBP and DNase remained to be elucidated. Effect of PRV DBP on the processivity of PRV DNase To obtain insight into the mechanism by which DBP stimulates the DNase, we performed the competition experiments. The labeled DNA (0:5 lg) was incubated with DNase and/or DBP, and at the logarithmic phase of reaction (the 2nd min), a 50-fold excess of unlabeled DNA was added to the reaction. The radioactive counts in the acid-soluble products were determined at each time point after chasing with unlabeled substrate. Because PRV DBP enhanced the PRV DNase activity, we used 0.5 pmol of PRV DNase instead of 0.75 pmol of PRV DNase in the presence of DBP. Under these conditions, the similar slope could be observed in both Figs. 6A and B (open squares). If the enzyme is unable to
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Fig. 3. Time course of PRV DBP on the exonuclease activity of PRV DNase. Purified PRV DNase (0.5 pmol) was mixed with 0:5 lg linear pUC18 dsDNA in the absence (A) or presence (B) of 10 pmol PRV DBP. Purified PRV DBP (10 pmol) was mixed alone with 0:5 lg linear pUC18 dsDNA (C). The mixtures were then incubated at 37 °C for 0 (lane 1), 1 (lane 2), 3 (lane 3), 5 (lane 4), 10 (lane 5), 15 (lane 6), or 20 (lane 7) min, and analyzed by 1.2% agarose gels. (D) Quantitative analysis of DNase activity in the absence or presence of PRV DBP. The amount of linear DNA was quantitated by Phoretix 1D program. Relative DNase activity was calculated by dividing the amount of DNase-treated DNA by the amount of DNase and DBP-treated DNA.
Fig. 4. Effect of acetic anhydride on the DNA-binding ability of PRV DBP. Purified PRV DBP was modified with various moles of acetic anhydride at 4 °C for 30 min. The DNA-binding ability of modified DBP was analyzed by nitrocellulose filter binding assay.
detach from the substrate once hydrolysis has begun, there should be no change in the rate of release of radioactivity. Alternatively, for an enzyme with a nonprocessive mode (distributive mode), the rate will decrease after engaging on the 50-fold excess of cold substrate. Addition of excess challenger DNA to ongoing DNase reaction in the absence of DBP showed no change in DNA hydrolysis (Fig. 6A), suggested that PRV DNase behaved in a processive mode. However,
Fig. 5. Effect of DNA-binding ability of PRV DBP on the PRV DNase activity. Purified DNase (0.5 pmol) was mixed with 50 ng 35 S-labeled pUC18 dsDNA in the presence of 10 pmol DBP, 10 pmol acetic anhydride-modified DBP, or 10 pmol of acetic anhydride-treated E. coli SSB. The mixture was incubated at 37 °C for 15 min, the undigested DNA was precipitated by TCA, and the radioactivity of acid-soluble nucleotides was determined with the scintillation counter. Relative DNase activity was calculated by dividing the radioactivity of DNasetreated DNA by the radioactivity of DNase and DBP-treated DNA.
the PRV DNase was efficiently competed from the DNA substrate in the presence of DBP (Fig. 6B), implied that PRV DBP stimulated the dissociation of DNase from DNA substrate and therefore increased its distribution.
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Fig. 6. Effect of PRV DBP on the processivity of PRV DNase. Purified PRV DNase (0.75 or 0.5 pmol, respectively) was mixed with 0:5 lg of 35 S-labeled pUC18 dsDNA in the absence (A) or presence (B) of 10 pmol DBP. After a 2-min incubation at 37 °C, a 50-fold excess of unlabeled pUC18 dsDNA was added and incubated at 37 °C for 0, 2, 10, 20, 40, or 60 min (filled squares). The undigested DNA was precipitated by TCA and the radioactivity of acid-soluble nucleotides was determined by scintillation counter. Open squares indicate control reaction (no added unlabeled pUC18 dsDNA). The arrow indicates the addition of unlabeled pUC18 dsDNA.
Physical interaction between PRV DBP and DNase The functional interaction between DNase and DBP was observed in this study. To further analyze whether the DNase activity stimulated by DBP was due to direct physical interaction, we carried out the ELISA experiment. Purified DNase or DBP was adsorbed onto the wells of plastic microtiter plates and blocked the remainder of the free surface with BSA. We challenged the immobilized DNase or DBP with various amounts of purified DBP or DNase, respectively. The DBP or DNase, bound to the DNase- or DBP-coated wells, was quantitated with respective primary Ab and peroxidaseconjugated goat anti-mouse IgG (Fig. 7). The interactions between immobilized DNase and DBP with respective Abs were served as positive controls (column 1), whereas the interactions between immobilized DNase and DBP with anti-DBP Ab and anti-DNase Ab, respectively, were used as negative controls (column 2). Previous result showed the specific interaction between
Fig. 7. Physical interaction between PRV DBP and DNase by ELISA. PRV DNase (open bar) or DBP (close bar) was immobilized on the plastic surfaces of the wells of microtiter plates and was challenged with various amounts of DBP or DNase, respectively. The positive controls (column 1) represent the interaction between immobilized DNase or DBP with respective Ab. The negative controls (column 2) represent the interaction between immobilized DNase or DBP with anti-DBP or anti-DNase Ab, respectively. Values are means standard error of triplicate assays.
PRV DNase and DBP, we therefore did not test the physical interaction between PRV DNase and another DNA-binding protein. The results showed that PRV DBP and DNase readily bound to the immobilized DNase and DBP, respectively, and the interaction between these proteins showed a dose-dependent manner. Interactions between herpesviral DNase and DBP have been reported in several studies. HSV-1 ICP8 forms a complex with alkaline nuclease and colocalizes within the nuclear replication compartments or prereplicative sites, suggested that these two proteins form a specific and probably functional complex [19,20]. EBV mDBP is complexed with DNase, and this complex is already present before the synthesis of viral DNA [26]. Our data, showing that PRV DNase physically interacts with PRV DBP by ELISA, are thus consisted with their results. Several studies suggested that herpesviral DBP and DNase play important roles in viral recombination [9,14,34]. Recombinational repair process consists of four steps in all organisms [35]. In E. coli, RecBCD enzyme binds to the end of a double-strand break and unwinds the dsDNA [36]. Upon recognizing the x sequences, the 30 ! 50 nuclease activity of RecBCD is attenuated, whereas a weaker 50 ! 30 activity is activated [37]. RecBCD then promotes the loading of RecA onto
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the x-containing ssDNA [38]. The resulting RecA protein-ssDNA filament invades homologous dsDNA to produce a D-loop structure and extends the DNA heteroduplex by branch migration [39]. Specific cooperation between x sites, RecBCD, RecA, and SSB may be a central theme in double-strand break repair by homologous recombination [40]. The x-sites switch the RecBCD polarity by attenuating the 30 ! 50 exonuclease activity of RecBCD and increasing the 50 ! 30 exonuclease activity [37]. RecBCD enzyme stimulates the preferential use of xfs-containing DNA by RecA protein [38]. SSB protein reduces the level of DNA degradation by RecBCD enzyme during unwinding [41]. Previous studies displayed that PRV DBP exhibited a base preference of dG and dC [27]. This study showed that PRV DBP stimulated the DNase activity via binding to dsDNA, and converted it into a non-processive enzyme. We therefore speculated that during replication, PRV DBP might bind to the GC-rich viral genome, promote the binding and cleavage of PRV DNase on the terminal repeated regions of viral genomes, and then stimulate the dissociation of the DNase from DNA substrate. The dissociation of DNase from DNA substrate increased the distribution of DNase to another viral genome and started another round of cleavage. The resulting terminal regions processed by DNase provided sources to anneal to complementary repeated regions and initiate the recombination event. The important question of whether the DNase activity was attenuated by terminal regions of viral genomes and then created the singlestranded end for recombination remained unresolved. Furthermore, in addition to PRV DNase and DBP, other proteins involved in viral recombination could not be excluded. We have now showed that functional interaction between PRV DBP and DNase. It should now be straightforward to examine further the biological meaning of this interaction.
Acknowledgments I thank Dr. Tin-Yun Ho and Dr. Ke-Jung Huang for a critical review of the manuscript. I thank Miss Wei-Yun Sheng for her technical assistance. This work was supported by grants from National Science Council and China Medical College, Taiwan.
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[25] P.V.A. Ratnakar, B.K. Mohanty, M. Lobert, D. Bastia, The replication initiator protein ð of the plasmid R6K specifically interacts with the host-encoded helicase DnaB, Proc. Natl. Acad. Sci. USA 93 (1996) 5522–5526. [26] Y. Zeng, J. Middeldorp, J.J. Madjar, T. Ooka, A major DNA binding protein encoded by BALF2 open reading frame of Epstein–Barr virus EBV forms a complex with other EBV DNAbinding proteins: DNAase, EA-D, and DNA polymerase, Virology 239 (1997) 285–295, doi: 10.1006/viro.1997.8891. [27] Wu, S.L., Ho, T.Y., Tsai, S.H., Sheng, W.Y., Hsiang, C.Y. DNAbinding ability and renaturation of complementary DNA strands by recombinant pseudorabies virus DNA-binding protein, submitted. [28] L.T. Doan, J.J. Toulme, C. Helene, Involvement of tryptophyl residues in the binding of model peptides and gene 32 protein from phage T4 to single-stranded DNA. A spectroscopic method for detection of tryptophan in the vicinity of nucleic acid bases, Biochemistry 23 (1984) 1202–1210. [29] M.I. Khamis, J.R. Casas-Finet, A.H. Maki, J.B. Murphy, J. Chase, Investigation of the role of individual tryptophan residues in the binding of E. coli single-stranded binding protein to singlestranded polynucleotides, J. Biol. Chem. 262 (1987) 10938–10945. [30] W.T. Ruyechan, J.W. Olson, Surface lysine and tyrosine residues are required for interaction of the major herpes simplex virus type1 DNA-binding protein with single stranded DNA, J. Virol. 66 (1992.) 6273–6279. [31] J.F. Riordan, B.L. Vallee, Acetylation, Meth. Enzymol. 11 (1967) 565. [32] M. Bhagwat, L.J. Hobbs, N.G. Nossal, The 50 -exonuclease activity of bacteriophage T4 RNase H is stimulated by the T4
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Pseudorabies virus DNA-binding protein stimulates the exonuclease activity and regulates the processivity of pseudorabies virus DNase Chien-Yun Hsiang* Department of Microbiology, China Medical College, 91 Hsueh-Shih Road, Taichung 404, Taiwan Received 16 April 2002
Abstract The pseudorabies virus (PRV) DNase is an alkaline exonuclease and endonuclease, which exhibits an Escherichia coli RecBCD-like catalytic function. The PRV DNA-binding protein (DBP) promotes the renaturation of complementary single strands of DNA, which is an essential function for recombinase. To investigate the functional and physical interactions between PRV DBP and DNase, these proteins were purified to homogeneity. PRV DBP stimulated the DNase activity, especially the exonuclease activity, in a dose-dependent fashion. Acetylation of DBP by acetic anhydride resulted in a loss of DNA-binding ability and a 60% inhibition of the DNase activity, suggesting that DNA-binding ability of PRV DBP was required for stimulating the DNase activity. PRV DNase behaved in a processive mode; however, it was converted into a distributive mode in the presence of DBP, implying that PRV DBP stimulated the dissociation of DNase from DNA substrates. The physical interaction between DBP and DNase was further analyzed by enzyme-linked immunosorbent assay, and a significant interaction was observed. Thus, these results suggested that PRV DBP interacted with PRV DNase and regulated the DNase activity in vitro. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Acetic anhydride; Chemical modification; DNase; DNA-binding protein; Endonuclease; Exonuclease; Physical interaction; Processivity; Pseudorabies virus; Recombination
Pseudorabies virus (PRV) is a member of Alphaherpesvirinae [1]. Alphaherpesviruses, such as herpes simplex virus type 1 (HSV-1), varicella-zoster virus, and PRV, have a closely similar gene arrangement and share a considerable amino acid sequence homology in their gene product [2]. During PRV DNA replication, seven proteins, including origin-binding protein, DNA polymerase/accessory complex, helicase/primase complex, and DNA-binding protein (DBP), are interacted and promote the rolling circle replication [3]. Additionally, a set of enzymes involved in DNA repair and nucleotide metabolism is expressed during viral replication. These enzymes are thymidine kinase, ribonucleotide reductase, deoxyuridine triphosphate nucleotidohydrolase, uracil glycosylase, and DNase [3].
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Fax: +886-4-2205-3764. E-mail address: [email protected] (C.-Y. Hsiang).
PRV DNase is a homolog of HSV-1 alkaline nuclease [4]. HSV-1 alkaline nuclease is not essential for DNA replication [5]. It is thought to provide nucleotides for HSV-1 DNA synthesis by degrading host cellular DNA [6]. It is also required for the efficient processing of viral DNA replication intermediates and for the egress of capsids from the nucleus [7,8]. Previous studies indicated that PRV DNase is able to cleave the double-stranded (ds) and single-stranded (ss) DNAs under the alkaline condition containing magnesium. Additionally, it exhibits an Escherichia coli RecBCD-like catalytic function with 50 -exonuclease, 30 -exonuclease, and endonuclease activities [9,10]. These and those results suggested that PRV DNase might play a role in the process of recombination and replication during PRV infection. PRV DBP is a homolog of HSV-1 infected cell polypeptide 8 (ICP8) [11]. ICP8 is essential for viral DNA synthesis [12]. It binds ssDNA rapidly and cooperatively [13]; it also promotes the renaturation of
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complementary single strands of DNA, which is an essential function of recombinase [14]. Several examples of physical and functional interactions between ICP8 and other enzymes have been described [15]. ICP8 interacts with the extreme C terminus of the origin-binding protein (UL9), greatly stimulating its DNA-dependent ATPase and DNA helicase activities [16]. Moreover, ICP8 stimulates the DNA polymerase/accessory complex [17] and assists the helicase/primase complex by preventing the reannealing of complementary single strands produced by its helicase action [18]. These results indicated that ICP8 might play an important role in the process of recombination and replication during herpesvirus infection. Since the HSV-1 ICP8 forms a complex with alkaline nuclease and colocalizes within the nuclear replication compartments [19,20], it is interesting to analyze the functional interaction between these two proteins in this study. Our data suggested that PRV DBP was associated with PRV DNase and this interaction might play an important role in viral recombination.
Materials and methods Proteins. Recombinant PRV DNase and DBP were expressed in E. coli BL21 (DE3) pLysS strain by transforming pET-DNase and pET/ DBP, respectively, to produce N-terminal fusions with six histidine residues. These proteins were purified to apparent homogeneity as described previously [9,11]. To ensure the correct folding of PRV DNase and DBP during purification, we carried out the DNase assay and nitrocellulose filter binding assay in each lot of proteins. The E. coli single-stranded DNA-binding protein (SSB) was purchased from United States Biological. DNase assay. The endonuclease and exonuclease activities of PRV DNase were assayed by incubating supercoiled and linear dsDNA, respectively, with DNase [9]. The 10-ll reaction mixtures, containing 0:5 lg of supercoiled pUC18 dsDNA or EcoRI-linearized pUC18 dsDNA, various amounts of PRV DNase, and DNase buffer (2 mM MgCl2 , 10 mM 2-mercaptoethanol, and 50 mM Tris/HCl, pH 9.0), were incubated at 37 °C for 5 min. The reactions were terminated by adding 1 ll of 10 stop buffer (50% glycerol, 1% SDS, 0.1% bromophenol blue, and 100 mM EDTA), analyzed by 1.2% agarose gels, and visualized by ethidium bromide. The amount of supercoiled or linear DNA banding on an agarose gel was quantitated by densitometric scanning. Lysine modification. The lysine was modified by acetic anhydride as described previously [21]. Purified DBP (0.1 ml) was mixed with an equal volume of saturated sodium acetate in DBP storage buffer (20% glycerol, 0.5 mM dithiothreitol, 0.2% NP-40, 10 lM ZnCl2 , 0.1 mM phenylmethylsulfonyl fluoride, and 50 mM Tris/HCl, pH 8.0). After the addition of 2 ll acetic anhydride, the mixture was incubated at 4°C for 30 min. Various amounts of acetic anhydride were then added and the mixture was incubated at 4 °C for another 30 min. Nitrocellulose filter binding assay. The formation of protein–DNA complexes was measured by using alkali-treated nitrocellulose filter as described previously [11]. Briefly, nitrocellulose filters (Hybond-C super; Amersham) were soaked in 0.5 M KOH for 20 min at room temperature, washed with distilled deionized water, and stored in 100 mM Tris/HCl (pH 7.6) at 4 °C. Neither ssDNA nor dsDNA was retained on the alkaline-treated filter, but protein–DNA complex was efficiently retained. The pUC18 dsDNA was labeled with [a-35 S]dATP by nick translation [22] and denatured to ssDNA by boiling for 5 min
and chilling on ice. The reaction mixture contained 100 ng of 35 S-labeled pUC18 ssDNA and various amounts of DBP in the DNAbinding buffer (1 mM EDTA, 150 mM NaCl, and 10 mM Tris/HCl, pH 7.5). A 100-ll sample of this mixture was incubated at 25 °C for 20 min and then applied to the alkali-treated nitrocellulose filters by using BioDot microfiltration units (Bio-Rad). The filters were washed twice with DNA-binding buffer and dried; the radioactivity retained on filters was determined with the scintillation counter (Beckman). Processivity of DNase. Processivity of DNase was performed as described previously [23]. PRV DNase (0.75 pmol) was mixed with 0:5 lg 35 S-labeled pUC18 dsDNA in 100 ll DNase buffer. After a 2-min incubation at 37 °C, a 50-fold excess of unlabeled pUC18 dsDNA was added and incubated at 37 °C as indicated. The undigested DNA was precipitated by adding 5 ll of 5 mg/ml bovine serum albumin (BSA) and 100 ll of 5% (w/v) trichloroacetic acid (TCA). The supernatant, containing acid-soluble nucleotides, was collected and the radioactivity of the nucleotides was determined with the scintillation counter (Beckman). Antibodies. The polyclonal antibodies (Abs) against DNase and DBP were prepared as described previously [24]. Briefly, Balb/c mice were injected with 0.5 ml pristane per mouse intraperitoneally. Two weeks later, 10 lg of protein was emulsified with an equal volume of Freund’s complete adjuvant (Gibco BRL) and injected subcutaneously. The protein boosted the mouse at a two-week interval twice. After the final immunization, 5 105 myeloma cells were injected intraperitoneally to each mouse, and the ascite, containing Abs, was collected daily for 5–8 days. Enzyme-linked immunosorbent assay (ELISA). The physical interaction between proteins was assayed by ELISA as described previously [25]. Microtiter plates (Costar) were incubated at 4 °C overnight with 50 ll of 10 ng=ll purified DBP or DNase, which was diluted in 0.05 M carbonate buffer (pH 9.6), as first layers. The wells were rinsed with 200 ll of washing buffer (0.5% Tween 20 in phosphate-buffered saline), and blocked with 200 ll blocking buffer (5% BSA in washing buffer) by incubating at 37 °C for 30 min. The absorbed protein in each well was challenged with various amounts of the second protein dissolved in 100 ll of blocking buffer and incubated at 37 °C for 1 h. The wells were then washed three times with washing buffer, and 50 ll of diluted mouse Ab, raised against the second protein, was added to each well and incubated at 37 °C for 1 h. After three washes with washing buffer, a 23,000-fold dilution of the goat anti-mouse IgG that was conjugated with peroxidase (Sigma) was added to each well and incubated at 37 °C for 1 h. Following three washes, 50 ll of the chromogenic substrate, 2; 20 -azinobis(3-ethylbenzthiazoline-sulfonic acid), was added to each well and incubated at 37 °C for 15 min. The absorbance was read at 405 nm in an ELISA plate reader.
Results and discussion Effect of PRV DBP on the PRV DNase activity Interactions between herpesviral DNase and other proteins have been reported in several studies [19,26]. To test whether the PRV DBP was able to influence the DNase activity, the exonuclease and endonuclease activities of PRV DNase were analyzed in the presence of DBP. The concentration of DNase used in this study was 0.5 pmol, which showed a limited DNase activity, and the DNase activity induced by DBP could be observed under this condition. Fig. 1 shows that the concentrations of DBP in excess of those of the PRV DNase (> 0:05 lM) stimulated the exonuclease activity of DNase up to 3.27-fold and this phenomenon displayed a dose-response manner. However, there was a slight
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Fig. 1. Effect of PRV DBP on the DNase activity. (A) Endonuclease and exonuclease activities of DNase induced by DBP. Lanes 1–5 represent the endonuclease activity by using supercoiled pUC18 dsDNA as the substrate. Lanes 6–10 represent the exonuclease activity by using linear pUC18 dsDNA as the substrate. Purified PRV DNase (0.5 pmol) was mixed with 0:5 lg dsDNA and 0 (lanes 2 and 7), 1 (lanes 3 and 8), 5 (lanes 4 and 9), or 10 (lanes 5 and 10) pmol of PRV DBP in the DNase buffer and incubated at 37 °C for 5 min. Lanes 1 and 6 contained no protein in the reaction. (B) Endonuclease and exonuclease activities of PRV DBP alone. Lanes 1–4 represent the endonuclease activity by using supercoiled pUC18 dsDNA as the substrate. Lanes 5–8 represent the exonuclease activity by using linear pUC18 dsDNA as the substrate. 0:5 lg dsDNA was mixed with 0 (lanes 1 and 5), 1 (lanes 2 and 6), 5 (lanes 3 and 7), or 10 (lanes 4 and 8) pmol of PRV DBP in the DNase buffer and incubated at 37 °C for 5 min. The resulting complex was analyzed by 1.2% agarose gels. Supercoiled and linear DNAs are indicated by arrows. (C) Quantitative analysis of DNase activity induced by PRV DBP. The amount of supercoiled or linear DNA was quantitated by Phoretix 1D program. Relative DNase activity was calculated by dividing the amount of DNase-treated DNA by the amount of DNase and DBP-treated DNA.
stimulation (1.48-fold) on the endonuclease activity in the presence of DBP. PRV DBP alone did not show the endonuclease and exonuclease activities (Fig. 1B). It showed a limited binding ability to dsDNA, which consisted of previous result [27]. To exclude the nonspecific stimulation of PRV DNase by another DNAbinding protein, we replaced the DBP with E. coli SSB and carried out the similar assay. No significant enhancement on both activities was observed (Fig. 2). Therefore, PRV DBP stimulated the exonuclease activity of PRV DNase, and this interaction was specific. To determine whether the stimulation of PRV DNase by DBP was due to an increase in the rate of DNA hydrolysis, time course experiments were carried out in the presence or absence of PRV DBP. The reaction mixtures, containing 0.5 pmol of PRV DNase, 0:5 lg linear DNA, and/or 10 pmol PRV DBP, were incubated
at 37 °C and stopped at indicated times. The concentration of DNase used in this study was 0.5 pmol, which showed a limited exonuclease activity, and the DNase activity induced by DBP could be observed under this condition. Fig. 3 shows that few DNAs were digested in the absence of DBP; however, the amount of digested DNA was increased in the presence of DBP as the incubation time was prolonged. DBP alone did not show the exonuclease activity. Therefore, PRV DBP displayed a stimulatory effect on the kinetics of DNA hydrolysis by PRV DNase. Effect of DNA-binding ability of PRV DBP on the DNase activity Basic and aromatic amino acid side chains have been shown to be involved in the interaction of prokaryotic
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Fig. 2. Effect of E. coli SSB on the PRV DNase activity. (A) Endonuclease activity. Purified PRV DNase (0.5 pmol) was mixed with 0.5 lg supercoiled pUC18 dsDNA and 0 (lane 1), 1 (lane 2), 5 (lane 3), or 10 (lane 4) pmol of E. coli SSB in DNase buffer and incubated at 37 °C for 5 min. (B) Exonuclease activity. Purified PRV DNase (0.5 pmol) was mixed with 0:5 lg linear pUC18 dsDNA and 0 (lane 1), 1 (lane 2), 5 (lane 3), or 10 (lane 4) pmol of E. coli SSB in DNase buffer and incubated at 37 °C for 5 min. The resulting complex was analyzed by 1.2% agarose gels. Supercoiled or linear DNA was indicated at the right. Lane C contained no protein in the reaction.
and eukaryotic single-stranded DNA-binding proteins with DNA [28–30]. To analyze whether the lysine residues were involved in DNA-binding ability of PRV DBP, and whether the DBP enhanced the DNase activity via binding to DNA, we carried out the chemical modification. The e-amino groups of lysine of PRV DBP were modified with acetic anhydrides. This reaction converts the positive charge of the amino groups to zero charge [31]. The DBP was treated with various amounts of acetic anhydride under alkaline conditions and the DNA-binding ability of DBP was evaluated by nitrocellulose filter binding assay. Fig. 4 shows that PRV DBP was acetylated by acetic anhydride in a dosedependent manner. Additionally, the acetylation of DBP by acetic anhydride resulted in a loss of DNAbinding ability, compared with the control sample. Thus, these results suggested that lysine residues were required for the interaction between PRV DBP and DNA. The DNase activity was further analyzed in the presence of wild-type DBP, acetic anhydride-modified
DBP, or acetic anhydride-modified E. coli SSB. Wildtype DBP enhanced the DNase activity up to 5-fold. In contrast, acetic anhydride-modified DBP exhibited a 60% inhibition on the DNase activity (Fig. 5). Acetic anhydride-modified E. coli SSB did not influence the DNase activity, which indicated that the inhibition of DNase activity by acetic anhydride-treated DBP was not caused by the residual acetic anhydride. Thus, these results suggested that DNA-binding ability of PRV DBP was required for stimulating the DNase activity. PRV DBP enhanced the PRV DNase activity. The lysine-modified DBP lost not only its DNA-binding ability, but also its ability to stimulate DNase activity, which suggested that DNA-binding ability of PRV DBP was important for stimulating the PRV DNase activity. Additionally, it also indicated that lysine residues might be involved in DNA-binding ability of PRV DBP and the functional interaction between PRV DBP and DNase. The N-terminally truncated Epstein–Barr virus (EBV) major DNA-binding protein (mDBP), which lost its DNA-binding ability, inhibits the DNase activity in vitro [23]. The exonuclease activity encoded by EBV DNase is also inhibited when this enzyme is associated in vivo with mDBP [26]. These studies suggested that the inhibition of DNase activity by mDBP might be a kind of viral self-protective mechanism to protect the viral DNA from viral DNase attack during the productive viral DNA synthesis. Bacteriophage T4 SSB strongly stimulates the exonuclease activity of T4 RNase H and converts it into a non-processive nuclease, suggesting that T4 SSB controls T4 RNase H degradation of RNA primers and adjacent DNA during each lagging strand cycle [32]. HSV-1 UL9 protein and ICP8 form a tight complex and this interaction greatly stimulates the rate and extent of DNA unwinding catalyzed by the UL9 protein [33]. This study showed the functional and physical interactions between PRV DBP and DNase. However, the biological role of functional interaction between PRV DBP and DNase remained to be elucidated. Effect of PRV DBP on the processivity of PRV DNase To obtain insight into the mechanism by which DBP stimulates the DNase, we performed the competition experiments. The labeled DNA (0:5 lg) was incubated with DNase and/or DBP, and at the logarithmic phase of reaction (the 2nd min), a 50-fold excess of unlabeled DNA was added to the reaction. The radioactive counts in the acid-soluble products were determined at each time point after chasing with unlabeled substrate. Because PRV DBP enhanced the PRV DNase activity, we used 0.5 pmol of PRV DNase instead of 0.75 pmol of PRV DNase in the presence of DBP. Under these conditions, the similar slope could be observed in both Figs. 6A and B (open squares). If the enzyme is unable to
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Fig. 3. Time course of PRV DBP on the exonuclease activity of PRV DNase. Purified PRV DNase (0.5 pmol) was mixed with 0:5 lg linear pUC18 dsDNA in the absence (A) or presence (B) of 10 pmol PRV DBP. Purified PRV DBP (10 pmol) was mixed alone with 0:5 lg linear pUC18 dsDNA (C). The mixtures were then incubated at 37 °C for 0 (lane 1), 1 (lane 2), 3 (lane 3), 5 (lane 4), 10 (lane 5), 15 (lane 6), or 20 (lane 7) min, and analyzed by 1.2% agarose gels. (D) Quantitative analysis of DNase activity in the absence or presence of PRV DBP. The amount of linear DNA was quantitated by Phoretix 1D program. Relative DNase activity was calculated by dividing the amount of DNase-treated DNA by the amount of DNase and DBP-treated DNA.
Fig. 4. Effect of acetic anhydride on the DNA-binding ability of PRV DBP. Purified PRV DBP was modified with various moles of acetic anhydride at 4 °C for 30 min. The DNA-binding ability of modified DBP was analyzed by nitrocellulose filter binding assay.
detach from the substrate once hydrolysis has begun, there should be no change in the rate of release of radioactivity. Alternatively, for an enzyme with a nonprocessive mode (distributive mode), the rate will decrease after engaging on the 50-fold excess of cold substrate. Addition of excess challenger DNA to ongoing DNase reaction in the absence of DBP showed no change in DNA hydrolysis (Fig. 6A), suggested that PRV DNase behaved in a processive mode. However,
Fig. 5. Effect of DNA-binding ability of PRV DBP on the PRV DNase activity. Purified DNase (0.5 pmol) was mixed with 50 ng 35 S-labeled pUC18 dsDNA in the presence of 10 pmol DBP, 10 pmol acetic anhydride-modified DBP, or 10 pmol of acetic anhydride-treated E. coli SSB. The mixture was incubated at 37 °C for 15 min, the undigested DNA was precipitated by TCA, and the radioactivity of acid-soluble nucleotides was determined with the scintillation counter. Relative DNase activity was calculated by dividing the radioactivity of DNasetreated DNA by the radioactivity of DNase and DBP-treated DNA.
the PRV DNase was efficiently competed from the DNA substrate in the presence of DBP (Fig. 6B), implied that PRV DBP stimulated the dissociation of DNase from DNA substrate and therefore increased its distribution.
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Fig. 6. Effect of PRV DBP on the processivity of PRV DNase. Purified PRV DNase (0.75 or 0.5 pmol, respectively) was mixed with 0:5 lg of 35 S-labeled pUC18 dsDNA in the absence (A) or presence (B) of 10 pmol DBP. After a 2-min incubation at 37 °C, a 50-fold excess of unlabeled pUC18 dsDNA was added and incubated at 37 °C for 0, 2, 10, 20, 40, or 60 min (filled squares). The undigested DNA was precipitated by TCA and the radioactivity of acid-soluble nucleotides was determined by scintillation counter. Open squares indicate control reaction (no added unlabeled pUC18 dsDNA). The arrow indicates the addition of unlabeled pUC18 dsDNA.
Physical interaction between PRV DBP and DNase The functional interaction between DNase and DBP was observed in this study. To further analyze whether the DNase activity stimulated by DBP was due to direct physical interaction, we carried out the ELISA experiment. Purified DNase or DBP was adsorbed onto the wells of plastic microtiter plates and blocked the remainder of the free surface with BSA. We challenged the immobilized DNase or DBP with various amounts of purified DBP or DNase, respectively. The DBP or DNase, bound to the DNase- or DBP-coated wells, was quantitated with respective primary Ab and peroxidaseconjugated goat anti-mouse IgG (Fig. 7). The interactions between immobilized DNase and DBP with respective Abs were served as positive controls (column 1), whereas the interactions between immobilized DNase and DBP with anti-DBP Ab and anti-DNase Ab, respectively, were used as negative controls (column 2). Previous result showed the specific interaction between
Fig. 7. Physical interaction between PRV DBP and DNase by ELISA. PRV DNase (open bar) or DBP (close bar) was immobilized on the plastic surfaces of the wells of microtiter plates and was challenged with various amounts of DBP or DNase, respectively. The positive controls (column 1) represent the interaction between immobilized DNase or DBP with respective Ab. The negative controls (column 2) represent the interaction between immobilized DNase or DBP with anti-DBP or anti-DNase Ab, respectively. Values are means standard error of triplicate assays.
PRV DNase and DBP, we therefore did not test the physical interaction between PRV DNase and another DNA-binding protein. The results showed that PRV DBP and DNase readily bound to the immobilized DNase and DBP, respectively, and the interaction between these proteins showed a dose-dependent manner. Interactions between herpesviral DNase and DBP have been reported in several studies. HSV-1 ICP8 forms a complex with alkaline nuclease and colocalizes within the nuclear replication compartments or prereplicative sites, suggested that these two proteins form a specific and probably functional complex [19,20]. EBV mDBP is complexed with DNase, and this complex is already present before the synthesis of viral DNA [26]. Our data, showing that PRV DNase physically interacts with PRV DBP by ELISA, are thus consisted with their results. Several studies suggested that herpesviral DBP and DNase play important roles in viral recombination [9,14,34]. Recombinational repair process consists of four steps in all organisms [35]. In E. coli, RecBCD enzyme binds to the end of a double-strand break and unwinds the dsDNA [36]. Upon recognizing the x sequences, the 30 ! 50 nuclease activity of RecBCD is attenuated, whereas a weaker 50 ! 30 activity is activated [37]. RecBCD then promotes the loading of RecA onto
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the x-containing ssDNA [38]. The resulting RecA protein-ssDNA filament invades homologous dsDNA to produce a D-loop structure and extends the DNA heteroduplex by branch migration [39]. Specific cooperation between x sites, RecBCD, RecA, and SSB may be a central theme in double-strand break repair by homologous recombination [40]. The x-sites switch the RecBCD polarity by attenuating the 30 ! 50 exonuclease activity of RecBCD and increasing the 50 ! 30 exonuclease activity [37]. RecBCD enzyme stimulates the preferential use of xfs-containing DNA by RecA protein [38]. SSB protein reduces the level of DNA degradation by RecBCD enzyme during unwinding [41]. Previous studies displayed that PRV DBP exhibited a base preference of dG and dC [27]. This study showed that PRV DBP stimulated the DNase activity via binding to dsDNA, and converted it into a non-processive enzyme. We therefore speculated that during replication, PRV DBP might bind to the GC-rich viral genome, promote the binding and cleavage of PRV DNase on the terminal repeated regions of viral genomes, and then stimulate the dissociation of the DNase from DNA substrate. The dissociation of DNase from DNA substrate increased the distribution of DNase to another viral genome and started another round of cleavage. The resulting terminal regions processed by DNase provided sources to anneal to complementary repeated regions and initiate the recombination event. The important question of whether the DNase activity was attenuated by terminal regions of viral genomes and then created the singlestranded end for recombination remained unresolved. Furthermore, in addition to PRV DNase and DBP, other proteins involved in viral recombination could not be excluded. We have now showed that functional interaction between PRV DBP and DNase. It should now be straightforward to examine further the biological meaning of this interaction.
Acknowledgments I thank Dr. Tin-Yun Ho and Dr. Ke-Jung Huang for a critical review of the manuscript. I thank Miss Wei-Yun Sheng for her technical assistance. This work was supported by grants from National Science Council and China Medical College, Taiwan.
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