Tobacco proliferating cell nuclear antigen binds directly and stimulates both activity and processivity of ddNTP-sensitive mungbean DNA polymerase

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ABB Archives of Biochemistry and Biophysics 468 (2007) 22–31 www.elsevier.com/locate/yabbi

Tobacco proliferating cell nuclear antigen binds directly and stimulates both activity and processivity of ddNTP-sensitive mungbean DNA polymerase Sujit Roy a, Swarup Roy Choudhury a, Sunil K. Mukherjee b, Dibyendu N. Sengupta b

a,*

a Department of Botany, Bose Institute, 93/1 Acharya Prafulla Chandra Road, Kolkata 700-009, West Bengal, India International Centre for Genetic Engineering and Biotechnology, P.O. Box-10504, Aruna Asaf Ali Marg, New Delhi 110067, India

Received 13 August 2007, and in revised form 10 September 2007 Available online 19 September 2007

Abstract PCNA is well known as a component of DNA replication system and plays important roles in multiple cellular pathways in addition to replication and repair. In this work we have demonstrated the physical and functional interaction between tobacco PCNA and mungbean ddNTP-sensitive DNA polymerase which shares many physicochemical properties with family X-DNA polymerases except with the moderately processive mode of nucleotide incorporation. We have shown here that recombinant PCNA binds directly to mungbean DNA polymerase as revealed in affinity chromatography, pull-down and co-immunoprecipitation approaches. In vitro DNA polymerase activity assay and processivity analyses indicated recombinant PCNA specifically stimulates both activity and processivity of mungbean DNA polymerase. These observations lead to interesting speculation about the functional significance of the ddNTP-sensitive enzyme in replication event in higher plants since the enzyme has been shown to be active and expressed at an elevated level during the endoreduplication stages in developing mungbean seeds.  2007 Elsevier Inc. All rights reserved. Keywords: PCNA; Mungbean DNA polymerase; Processivity; Family X-DNA polymerase; DNA pol b; DNA pol k

Proliferating cell nuclear antigen (PCNA)1 has received considerable attention in recent years because of its role in multiple cellular pathways. In addition to DNA replication and repair events like base excision, it is also involved in various other processes including nucleotide excision repair [1], mismatch repair [2], cell cycle control [3], apoptosis [4] and transcription [5]. Thus PCNA has been termed as ‘‘cellular communicator’’ because of its ability to connect and coordinate several cellular processes. Structurally PCNA is a homotrimeric ring-shaped protein and acts as a sliding clamp for processive DNA synthesis by formation of trimeric ring that encircles the DNA template [6]. The primary amino acid sequences of PCNA *

Corresponding author. Fax: +91 033 2355388. E-mail address: dn_sengupta@rediffmail.com (D.N. Sengupta). 1 Abbreviations used: PCNA, proliferating cell nuclear antigen; IPTG, isopropyl-thio-b-D-galactopyranoside. 0003-9861/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.09.009

are highly conserved across the eukaryotic kingdom and more than 90% homology has been observed among plant PCNA proteins [7]. PCNA interacts with a variety of cellular proteins, which include the DNA replication factors, the DNA repair proteins and the cell cycle regulatory factors [8]. The interaction of PCNA with other protein is mediated by a sequence motif called PIM (PCNA interacting motif) present in a plethora of proteins, while PCNA also interacts with many other proteins that lack the typical PIM. Significant progress has been made in recent years in understanding the mechanisms of replication and repair of DNA damages in plants [9]. Recently the identification and characterization of DNA pol k has been reported from rice plant (Oryza sativa, L. cv. Nipponbare). Based on the genome database resources of rice and Arabidopsis thaliana, it has been suggested that pol k may be the only member of X-family DNA polymerase in higher plants and may

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substitute DNA pol b to carry out DNA repair and recombination. Furthermore, rice pol k showed distinct interaction with PCNA, which increased the processivity of the enzyme [10]. Recently we have reported the characterization of a 62-kDa single polypeptide ddNTP-sensitive DNA polymerase from the developing seeds of mungbean [11] where we observed an enhanced expression and activity of the enzyme particularly during the 16–18 days after fertilization stages where >70% nuclear DNA endoreduplication has previously been reported [12]. The purified enzyme showed close similarity in many important biochemical properties with mammalian DNA pol b and with rice DNA pol k, which are well-studied members of family X-DNA polymerase from animal and plant system. We also observed a significant level of homology of the N-terminal heptapeptide sequence of mungbean DNA pol with other known X-family members which are considered to be evolutionarily conserved single subunit enzyme with the ability to fill short gaps and devoid of 3 0 –5 0 exonuclease activity. Based on these, we argued for the assignment of mungbean DNA pol under X-family DNA polymerase in higher plant genome. However, mungbean DNA pol showed a characteristic moderately processive mode of DNA synthesis in vitro in contrast to the distributive synthesis by the X-family DNA polymerases and showed an increased activity and expression level during the endoreduplication stages in the developing seeds, suggesting a functional relevance of the enzyme in replication events [11]. Since PCNA is a well-known component of the DNA replication machinery and mungbean DNA polymerase activity is associated with the stages of developing seeds, we evaluated the question of potential interaction between these two proteins in vitro. In this study, we overexpressed the tobacco PCNA protein in the form of GST-fusion protein. We observed that purified recombinant PCNA binds directly to mungbean DNA polymerase to form complex in vitro as revealed in pull-down assay and immunoprecipitation experiments. We also observed that PCNA specifically and significantly stimulates both activity and processivity of mungbean DNA polymerase in vitro. Thus our observation illustrates a probable interaction between PCNA, which serves as a processivity factor in DNA replication and a ddNTP-sensitive DNA pol which shares similarity with family-X DNA pols and at the same time probably involved in the DNA replication events in developing seeds of mungbean with a moderate processivity. Materials and methods Tobacco PCNA cDNA was a generous gift from Prof. Sunil K. Mukherjee, ICGEB, New Delhi. Escherichia coli strain BL21 was from Ahersham Biosciences, USA. Restriction protease thrombin was purchased from Qiagen (USA). Ampicillin, lysozyme, leupeptin, deoxycholic acid, PMSF, IPTG and all other chemicals were purchased from Sigma (St. Louis, MO). DNase 1 and T4 PNK were obtained from Roche (Germany). M13 mp 18 (ss) DNA, dNTPs, ddNTPs, poly(dA)/oligo(dT),

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activated calf thymus DNA, Glutathione–Sepharose 4B, reduced glutathione and PVDF (Hibond-P) membrane were obtained from Amersham Pharmacia Biotech (USA). HPLC purified 17-mer M13 sequencing primer was obtained from Sigma Genosys (USA). [3H]dTTP (Sp. Ac. 89 Ci/ mmol) was from New England Nuclear (NEM) Dupont, USA. [c-32p]ATP (Sp. Ac. >4000 Ci/mmol) was obtained from BRIT, India. Polyclonal antibody (affinity purified IgG) generated against rat DNA polymerase b was a generous gift from Dr. S.H. Wilson, NIESH, USA. Anti-GSTPCNA antibody was obtained from Prof. Sunil K. Mukherjee, ICGEB, New Delhi. Anti-rabbit GST antibody and goat anti-rabbit IgG (APconjugated) were purchased from Bangalore Genei, India.

Expression and purification of recombinant tobacco PCNA The 62-kDa GST-PCNA fusion protein was overexpressed in E. coli BL21 host strain harbouring pGEX4T-1: PCNA. 1.0 kb tobacco cDNA was introduced in pGEX4T-1 expression vector under the control of tac promoter of GST which resulted in the expression of 62 kDa fusion protein (26-kDa GST with 36-kDa tobacco PCNA). The expression of the fusion protein was achieved with the addition of 1 mM IPTG and considerable amount of fusion protein was accumulated after 4 h of induction, which reached a plateau after the next 2 h of induction. Thus 4 h of induction with 1 mM IPTG concentration was taken as the optimal condition for the induction of the fusion protein. E. coli BL21 cells (harbouring the pGEX 4T-1-tobacco PCNA) were grown overnight in 10 ml of LB medium (containing 0.1 mg/ml ampicillin) at 37 C shaker incubator. One hundred microlitres of the overnight culture was inoculated into 10 ml of fresh LB medium containing 0.1 mg/ml ampicillin and incubated at 37 C shaker incubator for 2 h until the A600 had reached 0.5. Isopropyl-thio-b-D-galactopyranoside (IPTG) was added to the culture to a final concentration of 1 mM and grown at 37 C for 4 h. The cells were harvested at 5000g for 5 min, 4 C. Cell pellet was washed once with 1· PBS and resuspended in 1.0 ml of 50 mM Tris–Cl, pH 8.0, containing 1 mM PMSF. Lysozyme (10 mg/ml) was added to a final conc. of 0.2 mg/ ml. Tubes were kept at 4 C for 30 min with occasional stirring. Deoxycholic acid was then added at final concentration of 1 mg/ml and mixed properly by stirring with a microtip. Tubes were then incubated at 37 C for about 30 min. DNaseI was added (final 1 mg/ml) and incubated at room temperature for anther 30 min. It was then centrifuged at 12,000g for 10 min at 4 C. Pellet was resuspended in 400 ll buffer A containing 50 mM Tris–Cl, pH 8.0, 50 mM NaCl, 10 lg/ml Leupeptin and 1 mM PMSF. It was sonicated by 8–10 pulses of 10 s each with 30 s intervals with keeping the tubes on ice. The cell lysate was then centrifuged at 12,000g, 10 min, at 4 C. All the protein fractions were analyzed in 10% SDS– PAGE to monitor the solubility of the fusion protein in different fractions. We observed that sonication of the induced bacterial cells was more effective than lysozyme treatment (Fig. 1e, lanes 3 and 4) for solubilization of the fusion protein. Glutathione–Sepharose affinity resin was used to purify the fusion protein. Purification was performed from the soup after sonication. DTT was added to the soup to a final concentration of 1 mM. About 300 ll of pre-washed glutathione–Sepharose 4B resin was added to the sonicated soup and incubated at 4 C with gentle shaking for 45 min. The unbound protein was removed by centrifugation at 3000g for 5 min at 4 C. The resin was then washed four times with ice-cold PBS and finally bound protein was eluted from the resin by addition of 1 bed volume elution buffer containing 10 mM reduced glutathione in 50 mM Tris–Cl, pH 8.0. Protein fractions were analyzed in 10% SDS–PAGE followed by staining with Coomassie blue. A single purified band of the GST-PCNA fusion protein of 62-kDa size was obtained after purification without any other contaminating bands (Fig. 1f, lanes 3–6).

Preparation of nuclear extract Nuclear extract was prepared as described by Gupta et al. [13] with slight modifications. About 50 gm of freshly harvested mungbean seeds from 16 to 18 daf stages was homogenized in 1:3 volumes of ice-cold nuclei

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Fig. 1. Recombinant tobacco PCNA binds to ddNTP-sensitive DNA polymerase in nuclear extract. (a) Immunoblotting of elution fraction of recombinant PCNA-bound mungbean seed nuclear extract from 16 to 18 daf stages (lane 1) with anti-rat pol-b antiserum. 2.5 lg of 62-kDa GST-PCNA fusion protein (lane 2) and 26-kDa GST (lane 3) were used to reveal the specificity of the antibody. (b) The blot was striped and immunoprobed with antiGST-PCNA polyclonal antibody, which revealed a faint 62-kDa band of recombinant PCNA co-eluted with mungbean seed NE and another band of 30-kDa (lane 1). Lanes 2 and 3 show cross-reactive bands for recombinant PCNA (62-kDa) and GST (26-kDa). (c) Western blotting of the similar NE with anti-rat pol-b antiserum to verify the expression of the 62-kDa mungbean DNA polymerase at the indicated stage (lane 2). Mungbean seed NE from the indicated stage (400 ng) resolved by 10% SDS–PAGE and stained with Coomassie blue (lane 1). NE, nuclear extract; IP, immunoprobe; daf, days after fertilization. (d) In-vitro DNA polymerase activity carried out using 400 ng of mungbean seed NE from 16 to 18 daf stages in the absence or presence of 10 lM of ddTTP or 300 lM of aphidicolin. (e) Induction and purification of recombinant tobacco PCNA. Solubilization of the bacterially expressed fusion protein by lysozyme treatment (lane 3) and sonication (lane 4) of the 1 mM IPTG induced E. coli BL21 cells harbouring pGEX4T-1: tobacco PCNA, after incubation at 37 C for 4 h (lane 2). Lane 1 shows E. coli lysate without IPTG induction. (f) Purification of fusion protein by affinity elution from glutathione–Sepharose resin. Lane 1 is soup of the IPTG induced bacterial cells after sonication. Bound protein was eluted with 10 mM reduced glutathione in 50 mM Tris–Cl, pH 8.0 (lanes 2–6). Molecular weight markers are shown on the left of figures. isolation buffer (25 mM Tris–Cl, pH 7.5, 1 M Sucrose, 10 mM MgCl2, 1 mM 2-mercaptoethanol and 0.5% Triton X-100) at 4 C. The homogenized suspension was filtered through three layers of sterile muslin cloth while keeping the filtrate over ice. Triton X-100 was added drop wise to the filtrate to a final concentration of 0.5%. Homogenized sample was then kept on ice for 30 min with occasional stirring and centrifuged at 5000g for 5 0 at 4 C. The pellet was washed with ice-cold nuclei wash buffer (25 mM Tris–Cl, pH 7.5, 0.44 M sucrose, 10 mM MgCl2 and 1 mM 2-mercaptoethanol) for three times by centrifugation at 5000g for 10 min at 4 C. The nuclei were lysed in 15 ml lysis buffer (100 mM KCl, 15 mM Hepes– KOH, pH 7.8, 5 mM MgCl2, 1 mM EDTA, pH 8.0, 1 mM DTT, 1 mM PMSF, 5 lg/ml leupeptin and 1 lg/ml antipain) and kept on ice for 45 min with occasional shaking. About 3 (M) KCl was added drop wise to a final concentration of 485 mM with frequent stirring of the tubes and keeping

on ice in-between stirring. It was then centrifuged for 20 min at 15,000g at 4 C. The final supernatant was dialyzed against 1000 volumes of dialysis buffer (20 mM Hepes–KOH, pH 7.8, 10% glycerol, 40 mM KCl, 10 mM MgCl2, 1 mM 2-mercaptoethanol and 1 mM PMSF) at 4 C for overnight with 2–3 changes. Dialyzed extracts were lyophilized at 42 C for 2.5 h. Aliquots were stored at 70 C. Concentration of the nuclear protein extract was determined by Bradford method [14] followed by assay for DNA polymerase activity in the presence of different inhibitors.

GST-pull-down assay Purified mungbean DNA polymerase (1.0 lg) was incubated with equal amounts of purified recombinant PCNA proteins in binding buffer

S. Roy et al. / Archives of Biochemistry and Biophysics 468 (2007) 22–31 containing 25 mM Tris–Cl, pH 7.5, 10% glycerol, 75 mM NaCl, 2.5 mM EDTA, 5 mM MgCl2, 2.5 mM DTT and 0.1% NP-40 with rotation at 4 C for overnight following which 50 ll of pre-washed and binding buffer equilibrated glutathione–Sepharose resin was added. The mixture was slowly stirred at 4 C for 3 h. The unbound protein fraction was separated from the resin by centrifugation at 3000g for 3 min, 4 C. The resin containing the bound protein was washed with 200 ll of ice-cold binding buffer by centrifugation (3000g for 3 min, 4 C). Finally the bound proteins were eluted by 1 bed volume of elution buffer containing 20 mM reduced glutathione in 50 mM Tris–Cl, pH 8.0. Equal amounts of purified mungbean DNA polymerase and GST was incubated with glutathione beads as control. The protein fractions were analyzed in 12% SDS–PAGE and the protein bands were visualized by staining with silver nitrate.

Co-immunoprecipitation Co-immunoprecipitation of purified mungbean DNA polymerase and recombinant PCNA was performed by incubating equal amounts (1.5 lg) of proteins in binding buffer containing protease inhibitors (1 mM PMSF and 5 lg/ml leupeptin) for 4 h at 4 C with rotation. To this mixture 0.6 lg of anti-rat pol b polyclonal antibody was added and again incubated for 4 h at 4 C. The immunocomplex was adsorbed onto binding buffer equilibrated protein-A agarose. The mixture was incubated at 4 C for overnight with rotation. Beads were washed three times with binding buffer containing protease inhibitors and finally resuspended in SDS-sample buffer, heated for 5 min and separated in 12% SDS–PAGE. Proteins were then transferred onto PVDF membrane (Hybond-P, Amersham Pharmacia) and eventually probed with antitobacco PCNA polyclonal antibody (1:10,000 dilution). Goat anti-rabbit IgG conjugated with alkaline phosphatase (1:1000 dilution) was used as secondary antibody and immunoreactive bands on the membrane were detected by following the enzymatic assay for alkaline phosphatase for colour development as described in Sambrook et al. [15]. Similar blot was stripped by incubating with buffer containing 65 mM Tris–Cl, pH 6.8, 100 mM b-mercaptoethanol and 1% SDS for 30 min at 50 C, followed by two washes with TBS-T at room temperature. The presence of mungbean DNA polymerase was confirmed by incubating the blot with rat pol b polyclonal antibody (1:10,000 dilution). Similarly the purified proteins were immunoprecipitated with anti-tobacco GST-PCNA polyclonal antibody. The blot was developed with anti-pol b polyclonal antibody to detect mungbean DNA polymerase. After stripping the blot, presence of PCNA was confirmed using anti-tobacco PCNA polyclonal antibody.

Measurement of DNA polymerase activity To study the effect of purified PCNA in vitro on the activity of mungbean DNA pol, about 100 ng of purified mungbean DNA polymerase was pre-incubated with increasing amounts (1–5 lg) of purified GST-PCNA fusion protein and with purified GST alone at 4 C for 4 h with rotation. In vitro DNA polymerase activity assay was preformed by measuring the incorporation of [3H]dTMP in 10% trichloroacetic acid insoluble fractions by following the protocol as described previously [16]. Reactions were performed in a final volume of 50 ll containing 50 mM Tris–Cl, pH 7.5, 5 mM MgCl2, 10 mM DTT, 0.05 mM of each of dATP, dCTP, dGTP and 1 lCi of [3H]dTTP (Sp. Ac. 89 Ci/mmol) and 20 lg/ml of activated calf thymus DNA or poly(dA)-oligo(dT) as template–primer. Reaction mixtures were incubated at 37 C for 30 min and then terminated by the addition of 5 ll of carrier DNA (sheared salmon sperm DNA, 10 mg/ml) and 2 ml of chilled 10% TCA containing 100 mM sodium pyrophosphate. After proper mixing reactions were kept on ice for 30 min. The precipitates were collected on prewashed (with sterile H2O) GF/C filters (Whatman) in a vacuum filtration device (Millipore). Filters were then washed with ice-cold 2% TCA twice and once with 90% ethanol. Filters were dried under heat lamp and radioactivity of each filter was measured in a Liquid Scintillation Counter (Beckman L5) using scintillation fluid. One unit of Klenow was

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pre-incubated with increasing amounts of recombinant fusion protein as control.

Primer extension assay Influence of PCNA on the processivity of mungbean DNA pol was studied by monitoring the in vitro replication of M13 mp 18 ss DNA as template using 5 0 -[c-32p]ATP labeled 17-mer forward sequencing primer (5 0 -GTTTTCCCAGTCACGAC-3 0 ) as described before [17]. 20 pmol of the 17-mer oligo were used for end labeling with T4 polynucleotide kinase. 1.0 lg purified mungbean DNA pol was pre-incubated with increasing amounts of purified recombinant tobacco PCNA or GST and with rat pol b antibody for at 4 C for 4 h with rotation. The primer extension reaction was carried in 50 ll reaction mixture containing 50 mM Tris–Cl, pH 7.5, 5 mM MgCl2, 2 mM DTT, 20 mM NaCl, 200 lg/ml BSA, 2% glycerol, 280 lM of each dATP, dCTP, dGTP, dTTP and 200 fmol of hybridized template:primer. The reaction mixtures were incubated at 37 C for 10 min and terminated by addition of 20 mM EDTA. Radiolabeled products were purified by phenol:chloroform and finally dissolved in 10 ll of sequencing gel-loading buffer (10 mM EDTA, 0.3% bromophenol blue and 0.3% xylene cyanol in 97.5% formamide). The products were resolved by denaturing polyacrylamide gel electrophoresis (7 M urea and 8% polyacrylamide) and visualized after exposure to Kodak X-omat film.

Replication assays in the presence of a DNA trap Reaction mixtures containing 1.0 lg of purified mungbean DNA pol with or without PCNA were pre-incubated with the radiolabeled M13 template/primer complex in the standard reaction buffer in the absence of dNTPs at 37 C for 5 min to pre-form the substrate–polymerase complex. Enzymatic reactions were started by the addition of dNTPs (280 lM) or dNTPs and excess of competitor DNA (1 mg/ml of non-radiolabeled salmon sperm DNA as cold trap) to capture enzyme molecules that dissociated from the radio labeled template and then incubated at 37 C for 10 min.

Results and discussion Tobacco PCNA binds to mungbean ddNTP-sensitive DNA polymerase in nuclear extract To investigate the putative interaction between mungbean DNA polymerase and tobacco PCNA, 10 lg of E. coli cell lysate over-expressing the tobacco GST-PCNA fusion protein was immobilized in glutathione–Sepharose column and through which about 400 lg of mungbean seed nuclear extract from 16 to 18 daf stages was chromatographed. The loosely bound proteins were washed with ice-cold PBS buffer and then the firmly bound proteins were eluted with 20 mM reduced glutathione in 50 mM Tris–Cl, pH 8.0. Elution fractions were subjected to protein gel blot analysis using anti-rat b pol antiserum (Fig. 1a). Since mungbean DNA pol has shown close immunological similarity with mammalian DNA pol b and anti-rat DNA pol b antibody specifically recognizes mungbean DNA pol [11], we used the anti b pol antiserum to detect the presence of mungbean DNA pol in the elution fractions. A distinct cross reactive band of 62 kDa was obtained in the column eluted fraction (Fig. 1a, lane 1) while the absence of any immunoreactive band in the lanes containing purified GST-PCNA and GST (lanes 2 and 3) in the similar blot clearly indicating the specificity of antibody binding and

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the presence of mungbean DNA polymerase in the column elute. The similar protein samples were then immunoprobed with anti-GST-PCNA antiserum to detect the presence of bound recombinant PCNA in the elution fraction of the resin bound mungbean seed nuclear extract. Such analysis recognized the recombinant PCNA (62-kDa) in the elution fraction (Fig. 1b, lane 1) and the purified GST-PCNA or GST (26-kDa) (as control) in the same blot (lanes 2 and 3). A cross-reactive band at 30-kDa position was observed in the elution fraction of PCNA-bound nuclear extract, which probably correspond to mungbean nuclear PCNA as recognized by the tobacco PCNA antiserum (Fig. 1b, lane 1). Since plant PCNA proteins are extremely (>90%) homologous, tobacco PCNA may be functionally equivalent to PCNA of mungbean origin and thus the anti-tobacco GST-PCNA antibody recognized the PCNA proteins from both the sources with almost equal efficiencies. Western blotting of mungbean nuclear protein extracts using anti pol b antibody showed the expression of 62kDa mungbean DNA polymerase in the 16–18 daf stage seed nuclear protein extract. The expression of the protein was also consistent with the ddNTP-sensitive and aphidicolin insensitive DNA pol activity in the nuclear extract of the similar stage (Fig. 1c and d). Taken together these results clearly indicates the expression and activity of a 62-kDa ddNTP-sensitive DNA pol in the nuclear protein extracts of developing mungbean seeds at 16–18 daf stages and our data also indicated a probable physical interaction between mungbean DNA polymerase and tobacco PCNA which binds to the DNA polymerase in the nuclear extract in vitro as revealed in the experiments involving affinity chromatography and immunoblotting. Recombinant PCNA associates with purified mungbean DNA polymerase To further validate the direct binding of recombinant PCNA to mungbean DNA polymerase at the biochemical level, a pull-down approach was followed where equal amounts of purified mungbean DNA polymerase and recombinant tobacco PCNA (purified by affinity column) was incubated in binding buffer and subsequently reacted with glutathione–Sepharose beads. After separating the loosely bound proteins, bound proteins were eluted with 50 mM Tris–Cl, pH 8.0, containing 20 mM reduced glutathione. The unbound (flow through, FT) and eluted fractions were separated in 12% SDS–PAGE followed by staining the gel with silver nitrate. Purified GST was incubated with purified mungbean DNA polymerase as control and similarly adsorbed to the resin. Shown in Fig. 2a, purified mungbean DNA pol did not interact with GST and to the resin and thus came out in the flow through fraction along with some unbound GST (lane 1). While the remaining resin bound GST appeared within the initial eluted fractions (lane 2). This result clearly rules out any interaction of mungbean DNA polymerase with GST.

In case of elution of the affinity resin containing purified mungbean DNA pol and recombinant PCNA, while some amount (40–45%) of proteins was obtained in unbound fraction (Fig. 2b, lane 1), substantial amounts proteins bound to the affinity resin was also detected and a distinct band of 62-kDa was visible in glutathione beads and in all the affinity elution fractions (lanes 2–6). Since purified mungbean DNA polymerase and recombinant PCNA have more or less similar molecular mass (62-kDa) it was difficult to demonstrate the presence of both the proteins in the similar elution fractions by separating the proteins followed by staining the gel. To address this issue, the intensities of the 62-kDa bands obtained after elution of the resin containing purified mungbean DNA pol and recombinant PCNA were compared with that of single purified protein bands of mungbean DNA polymerase and recombinant PCNA (Fig. 2a, lanes 5, 6, 2 lg of each of the purified protein was loaded). Quantitative estimation of the band intensities by densitometric analyses have shown an altogether higher intensity of 62-kDa bands in elution fractions than the single purified bands of each of the protein alone (data not shown). Together these results clearly provide a clue that recombinant PCNA associates with mungbean DNA polymerase and coelutes from the resin after elution. Since it was difficult to get uniform amounts of the two interacting protein partners in the elution fractions, the comparative quantitation of the band intensities of the purified protein alone and in the elution fraction was not sufficient to draw a significant conclusion. Therefore, to further substantiate these results, the elution fractions from the resin presumably containing mungbean DNA pol and recombinant PCNA were transblotted onto PVDF membrane and eventually immunoprobed with anti-rat pol b (Fig. 2c) and anti-GST-PCNA polyclonal antibodies (Fig. 2d), respectively. Distinct cross-reactive bands of 62-kDa was obtained with both the antibodies, which clearly demonstrate the presence of both mungbean DNA pol and recombinant PCNA in affinity elution fractions. Recombinant PCNA forms immunoprecipitable complex with mungbean DNA polymerase To further verify the speculated interaction, we carried out co-immunoprecipitation analysis where purified mungbean DNA pol was incubated with purified recombinant PCNA in binding buffer and then immunoprecipitated with anti-rat pol b antibody. The immuno complex, after separation in 12% SDS–PAGE, was transferred to PVDF membrane and the presence of recombinant PCNA in the complex was confirmed by probing the blot with antiGST-PCNA polyclonal antibody which showed a distinct cross reactive band of 62-kDa (Fig. 2e, upper panel). To verify that mungbean DNA pol had been immunoprecipitated in the same complex, the blot was stripped and immunoblotted with anti-rat pol b antibody (Fig. 2e, lower panel). In a similar fashion purified mungbean DNA polymerase and recombinant tobacco PCNA was immunoprecipitated with

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Fig. 2. In vitro association of recombinant PCNA with mungbean DNA polymerase. (a) GST-pull-down assay. As a control 6 lg of purified GST was reacted with 1.0 lg of purified mungbean DNA pol. Lane 1 shows the unbound proteins. Lanes 2–4 illustrate affinity resin elution fractions. Lanes 5 and 6 correspond to 2 lg of purified DNA pol and recombinant tobacco PCNA. (b) Serial elution of affinity resin containing recombinant PCNA and DNA pol (lanes 3–6). Lane 1 shows the unbound fraction while lane 2 is the resin bound protein. Proteins in various chromatographic fractions were examined by 12% SDS–PAGE followed by staining with silver salt. FT, flow through. Elution fractions from lanes 3–6 were subjected to Western blotting with antipol b (c) or anti-GST-PCNA antibody (d) to confirm the presence of both the proteins. (e) Immunoprecipitation (IP) with anti-pol b antibody. Upper panel corresponds to immunoblotting (IB) with anti-GST-PCNA antibody to detect PCNA and lower panel is for anti-pol b antibody to detect mungbean DNA pol. (f) Immunoprecipitation with anti-GST-PCNA antibody. Upper panel is the immunoblotting with anti-pol b antibody to detect mungbean DNA pol and lower panel is for anti-GST-PCNA antibody to detect PCNA. Lane 1 is for pre-immune IgG control and lanes 2 and 3 duplicate experiments for all the panels. Each experiment was repeated three times.

anti-GST-PCNA polyclonal antibody following which the presence of both the proteins in the complex was confirmed by immunoblotting with the respective antibodies (Fig. 2f, upper and lower panels). Taken together these results clearly illustrate that recombinant PCNA directly interacts with mungbean DNA polymerase and forms a complex in vitro, which can be immunoprecipitated. PCNA stimulates the activity of mungbean DNA polymerase To further analyze the functional significance of the putative interaction between tobacco PCNA with mungbean DNA polymerase, we have investigated the effect of PCNA on the activity of mungbean DNA polymerase in vitro. Affinity purified desalted fusion protein was used

in these experiments since thrombin digestion was not complete and the products of the digestion were virtually a mixture of fusion protein, PCNA, thrombin and GST. Complete separation of the PCNA from other protein proved unsuccessful. Since the effect of PCNA is best observed using a template with large gap, the influence of PCNA on mungbean DNA pol activity was studied using pol(dA)/oligo(dT)10–18 template–primer (Fig. 3a). Pre-incubation of the purified enzyme with increasing amounts of the recombinant PCNA (1–5 lg) showed distinct stimulation of activity which was optimal with 2–3 lg of fusion protein at both pH 7.5 and 6.8 (about 2-fold increase in activity than control). Although the activity stimulation was more distinct at pH 7.5 in comparison to pH 6.8, activity was distinctly

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Fig. 3. Effect of PCNA on the activity and processivity of mungbean DNA polymerase. (a) In vitro DNA pol activity assay was carried out after preincubation of 100 ng of purified enzyme in the presence of increasing amounts of recombinant PCNA or GST (1–5 lg). DNA pol activity was carried out at pH 7.5 and 6.8. Poly(dA)/oligo(dT) template was used at a final concentration of 20 lg/ml. As a control, 1 U of Klenow was pre-incubated with increasing amounts of PCNA followed by activity assay. (b) DNA pol was measured in the absence or presence of PCNA or GST using activated calf thymus DNA as template at pH 7.5 and 6.8. Klenow activity was measured in the presence of PCNA as control. (c) Shown is an illustration of annealing of 5 0 -[c-32p]ATP labeled forward sequencing primer (40) (17 nucleotides in length) to the M13 ss DNA template used in primer extension DNA synthesis for processivity analysis. (d) 8% sequencing gel image showing the effect of PCNA on processivity of mungbean DNA pol. Lane 1 shows processivity of the enzyme alone. Lanes 2–6 illustrate reaction products after pre-incubation of 1.0 lg of enzyme in the presence of 1–5 lg of PCNA. Lane 7 indicates reaction products in the presence of 3 lg of recombinant PCNA after pre-incubation of the DNA pol with 400 ng of an-rat pol b antibody. Lane 8 is the processivity in the presence of 5 lg of GST as control. (e) In vitro DNA pol activity carried out by monitoring the incorporation of [3H]dTMP using M13 primer/template after pre-incubation of mungbean DNA (100 ng of purified enzyme) with increasing amounts of recombinant PCNA or GST or reacting 1 U Klenow with increasing amounts of PCNA.

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inhibited with higher amount of fusion protein (5 lg). Activity of Klenow enzyme (E. coli DNA pol 1 large fragment, a ddNTP-sensitive enzyme used as positive control) after pre-incubation with similar amounts of fusion protein remain unaffected, while pre-incubation of mungbean DNA pol with increasing amounts of purified GST showed no stimulative or inhibitory effect. Thus it demonstrate that the recognition and activity stimulation of mungbean DNA polymerase by tobacco PCNA is very specific while excess amount of the fusion has an inhibitory effect. In addition, pH of the reaction plays an important function to affect such putative interaction. We have further analyzed the effect of recombinant PCNA on mungbean DNA pol activity by using activated calf thymus DNA template, the preferred template for mungbean DNA pol (Fig. 3b). Such analysis also indicated the stimulation of DNA pol activity by recombinant PCNA although the effect was much significant with the pol(dA)/oligo(dT)10–18 template. Taken together these results put on view a specific stimulation of mungbean DNA pol activity in the presence of recombinant PCNA in vitro under standard conditions. PCNA positively regulates the processivity of purified mungbean DNA polymerase Since PCNA has shown a distinct stimulatory effect on the activity of mungbean DNA pol, we then investigated whether PCNA has any effect on the mode of nucleotide incorporation by mungbean DNA pol. The influence of PCNA on the processivity of mungbean DNA polymerase was analyzed by primer extension DNA synthesis assay using the M13 mp 18 ss DNA template and 5 0 -[32p]-labeled 1-mer oligo (Fig. 3c) in the absence or presence of PCNA as described in Materials and methods. Processivity assay carried out without PCNA showed the usual moderately processive DNA synthesis with the incorporation of 35– 40 nucleotides after 10 min of incubation at 37 C (Fig. 3d, lane 1). This clearly indicated a PCNA independent processivity in contrast to pol d, which showed a PCNA dependent processivity [18]. On the other hand, a distinct stimulation of processivity was observed with 2– 4 lg of recombinant PCNA (lanes 3–5) after pre-incubation of purified DNA pol with increasing amounts of recombinant PCNA. Both the length of the products formed and the intensity of the bands increased significantly in the presence of 2–4 lg of the recombinant PCNA which indicating a significant incorporation of labeled primer into the larger products and efficient DNA synthesis on single stranded template in vitro. Optimal processivity was observed with 3–4 lg (lanes 4 and 5) of recombinant PCNA with the products up to 50 nucleotides in lengths were observed. As like activity processivity was distinctly inhibited at higher amount of PCNA (lane 6), which indicated that excess amount of PCNA might have negative influence on activity and processivity. Pre-incubation of the enzyme with rat pol b antibody distinctly inhibited pro-

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cessivity in the presence of 3 lg of recombinant PCNA (lane 7), thus indicating trapping of the enzyme by the antibody and thus bound enzyme was not accessible to carry out its normal processivity or interact properly with PCNA. Processivity remain unaffected after pre-incubation with purified GST (lane 8) illustrating the stimulation of processivity was entirely due to the putative interaction between the PCNA of the fusion protein and the DNA polymerase. Taken together, these results clearly demonstrate a specific stimulatory influence of PCNA on the processivity of the enzyme and that under appropriate assay conditions PCNA substantially increase the processivity of mungbean DNA pol. In vitro DNA pol activity assay using purified mungbean DNA polymerase and Klenow in the presence of increasing amounts of purified recombinant PCNA (1–5 lg) or GST (lg) in terms of incorporation of [3H]dTMP on M13 template–primer complex also indicated a specific stimulation of activity of mungbean DNA pol with 2–4 lg of recombinant PCNA (Fig. 3e). Only 1.1-fold increase in activity was obtained with 1 lg PNCA, while approximately 1.4-, 2.3and 2.2-fold stimulation of activity was obtained after pre incubation of the DNA pol with 2, 3 and 4 lg of PCNA, respectively, as compared to the control (without PCNA). About 1.7-fold decrease in activity was noticed with 5 lg of PCNA as compared with the activity obtained in the presence of 3 lg of PCNA. On the other hand, the activity of Klenow and mungbean DNA polymerase remained unaffected after pre-incubation with increasing amounts of PCNA and GST, but due to higher processivity, pmol of dNTPs incorporated by Klenow was high (2.7-fold higher) than that of mungbean DNA polymerase after incubation at 37 C for 30 min in the absence of PCNA. Taken together, these results demonstrate an efficient DNA replication in single stranded template and that PCNA probably help in stabilizing the template–DNA pol interaction to positively influence the activity and processivity on mungbean DNA pol as observed in vitro. Processivity in the presence of a molecular trap To validate whether the products of the processivity assay reflect DNA synthesis of single binding event, we studied the effect of PCNA on the processivity of mungbean DNA pol under conditions where the polymerase might only be expected to encounter a template/primer once. To address this, the reaction mixtures containing the DNA pol alone or with PCNA were pre-incubated at 37 C in the absence of dNTPs to pre-form a polymerase–DNA complex. The reactions were initiated by the addition of dNTPs (Fig. 4a, lanes 1 and 2) or dNTPs and an excess of competitor DNA (non-radiolabeled salmon sperm DNA) (Fig. 4a, lanes 4 and 5), which serves as a cold trap to capture DNA pol molecules as they dissociate from the template. Fig. 4a (lane 1 versus 2) shows the stimulation of pol processivity by PCNA under standard assay conditions where products of up to 50 nucleotides in

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Fig. 4. Processivity in the presence of a DNA trap. (a) Purified mungbean DNA pol (1.0 lg) alone or with 3 lg PCNA was incubated in a standard 50 ll reaction with 200 fmol of radiolabeled M13-primer/template at 37 C for 5 min and then reactions were initiated by the addition of dNTPs (280 lM) (lanes 1 and 2) or dNTPs and an excess of competitor DNA (1 mg/ml) to capture pol molecules after they dissociated from the radiolabeled primer–template (lanes 4 and 5). The reaction mixtures were then incubated at 37 C for 10 min. Lanes 1 and 2 display processivity without or with PCNA under standard conditions. In a control reaction, DNA pol and PCNA were pre-incubated with an excess of competitor DNA and labeled primer/template. dNTPs were then added and incubated at 37 C for 10 min (lane 3). Lanes 4 and 5 correspond to processivity where trap DNA was added after pre-incubation of the enzyme with labeled substrate without or with PCNA. (b) Influence of pH and divalent cation concentrations. Processivity assay carried out after preincubation of 1.0 lg of mungbean DNA pol with 3 lg of purified PCNA in the presence of increasing concentrations of Mg2+ (2, 4 and 6 mM, lanes 1–3) or without Mg2+ (lane 4) at pH 7.5. Similar analysis was performed in the presence of 0.1 and 1.0 mM Mn2+ (lanes 5 and 6). (c) Analysis of processivity after pre-incubation of the enzyme (1.0 lg) with PCNA (3 lg) in the absence (lane 1) or presence of 2, 4 and 6 mM Mg2+ (2–4) at pH 6.8.

lengths were obtained in the presence of 3 lg PCNA as opposed to the processivity of ±35–40 nucleotides by the DNA pol alone (Fig. 4a, lane 1 versus 2). Pre-incubation of the enzyme in the presence of an excess of competitor DNA resulted in the trapping of all pol molecules by the cold substrate and no polymerase activity was detected (Fig. 4a, lane 3) after the addition of dNTPs and PCNA. When competitor DNA was added after pre-incubation, mungbean DNA pol alone incorporated ±35–40 nucleotides (lane 4). In contrast, in the presence of PCNA up to 50 nucleotides were incorporated (lane 5). Taken together, these results clearly demonstrate after single encounter with the substrate the enzyme alone can incorporate up to 35– 40 nucleotides and up to 50 nucleotides in the presence of PCNA which specifically stimulate the processivity. Therefore the products of the processivity assay obtained in the absence or presence of PCNA indicate the true processivity under single hit condition and not resulted due to the repeated encounter of the enzyme with the already utilized substrate.

pH and divalent cation concentrations affect PCNA–DNA pol interaction To study the influence of pH and divalent cation concentrations on the putative interactions between mungbean DNA pol and PCNA, processivity assay was carried out after pre-incubation of DNA pol with PCNA under the conditions of varying pH and divalent cation concentrations. PCNA stimulated processivity of mungbean DNA pol was found to be reliant on reaction conditions like, pH, concentrations of divalent metal ions etc. Processivity assay was carried out after pre-incubation of 1.0 lg of purified mungbean DNA pol with 3 lg of recombinant PCNA (optimal amount of PCNA found to stimulate both activity and processivity of mungbean DNA pol) with varying concentrations of Mg2+ and Mn2+ ions at pH 7.5 (Fig. 4b). PCNA significantly stimulated the processivity in the presence of increasing concentrations of Mg2+ (2–6 mM, lanes 2–4). Both the length of the products and the intensity of the bands increased noticeably at 6 mM Mg2+ where the

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reaction products of up to >45 nucleotides in length were observed (lane 4). On the other hand no synthesis was observed in the absence of Mg2+ although PCNA was present in the reaction (lane 4). This clearly indicated the absolute requirement of Mg2+ for the activity of the enzyme and that PCNA stimulate the processivity when assay conditions are appropriate. Processivity of mungbean DNA pol showed a different behaviour in the presence of PCNA with increasing concentrations of Mn2+ than with Mg2+. Approximately 35–40 nucleotides were incorporated after pre-incubation of the enzyme with PCNA in the presence of 0.1 mM Mn2+ (lane 5), while processivity was strongly inhibited with 1.0 mM Mn2+ where incorporation up to <20 nucleotides were observed (lane 6). Further higher concentrations of Mn2+ beyond 1 mM drastically inhibited the processivity (not shown). We also studied the influence of PCNA on the processivity of the enzyme at lower pH, (pH 6.8) in the presence of varying concentrations of Mg2+ (Fig. 4c). Again no synthesis was observed in the absence of Mg2+ (lane 1). In contrast to pH 7.5, stimulation of processivity was optimal at 2 mM Mg2+ at lower pH (lane 2). Both the length of the products formed and the intensity of the bands decreased with increasing concentrations of Mg2+ (4 and 6 mM Mg2+, lanes 3 and 4). At low pH (6.8) increasing concentrations of Mn2+ showed inhibitory effect and no significant effect of PCNA on the processivity was observed even in the presence of 0.1 mM Mn2+ (not shown). Taken together, these results clearly demonstrate that both pH and concentrations of divalent cations play critical role to influence the putative interaction between mungbean DNA pol and PCNA. At higher pH, the interaction is more significant in the presence higher concentration of Mg2+. While the interaction is favoured by low concentration of Mg2+ at low pH. In addition, the enzyme showed a distinct preference for Mg2+ as a cofactor over Mn2+, which significantly inhibited the activity and thus processivity even at 1.0 mM concentration at both pH 7.5 and 6.8. In conclusion, our work illustrates a physical and functional interaction between recombinant tobacco PCNA and mungbean ddNTP-sensitive DNA polymerase which form immunoprecipitable complex in vitro. In addition our results have clearly indicated that although processivity of mungbean DNA pol was not dependent on PCNA but both activity and processivity of the enzyme was distinctly stimulated by PCNA in vitro under appropriate conditions. However, the efficiency of stimulation was much less as compared to the effect of PCNA on DNA pol d as observed in mammalian system. The probable reasons for this may be that mammalian DNA pol dII has a complete dependence on PCNA for the activity and processivity [18]. While purified mungbean DNA polymerase showed biochemical and immunological similarity with family-XDNA pols, which are well known for their repair activity rather that in replication and the moderate processivity

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of mungbean DNA pol did not show complete dependence on PCNA. Secondly, the large GST region in the fusion protein probably interfered in the physical interaction between the two proteins and thus nullifying the optimal stimulative effect. Finally it can be concluded that our results provide novel information on the interaction of a ddNTP-sensitive DNA polymerase with one of the most important component of the replication machinery, the PCNA in higher plant system. Further in vivo investigation on such interactions will shed new light into the understanding of molecular mechanism of DNA replication in plant cells. Acknowledgments We thank Dr. Samuel H. Wilson, NIEHS, Research Triangle, NC, USA, for the generous gift of rat DNA pol b polyclonal antibody. We also thank Mr. Jadav Ghosh, in-charge of Central Instrument Facility, Dept. of Botany, for providing the necessary technical support. The work is supported by the research grant from the Dept. of Botany, Bose Institute. Senior Research Fellowship to S.R. was provided by the Dept. of Botany, Bose Institute. S.R.C. is the recipient of Council of Scientific Industrial Research Fellowship [CSIR File No. 9/15 (283)/2003-EMR-1] from the Govt. of India. References [1] A.F. Nichols, A. Sanncar, Nucleic Acids Res. 20 (1992) 2441–2446. [2] A. Umar, A.B. Buermeyer, J.A. Simon, D.C. Thomas, A.B. Clark, R.M. Liskay, T.A. Kunkel, Cell 87 (1996) 65–73. [3] H. Watanabe, Z.Q. Pan, N. Schreiber-Agus, R.A. DePinho, J. Hurwitz, Y. Xiong, Proc. Natl. Acad. Sci. USA 95 (1998) 1392–1397. [4] M. Scott, P. Bonnefin, D. Vieyra, F.M. Boisvert, D. Young, D.P. Bazett-Jones, K. Riabowol, J. Cell Sci. 114 (2001) 3455–3462. [5] S. Hasan, P.O. Hassa, R. Imhof, M.O. Hottiger, Nature 410 (2001) 387–391. [6] J.M. Gulbis, Z. Kelman, J. Hurwitz, M. O’Donnell, J. Kuriyan, Cell 87 (1996) 297–306. [7] B. Bagewadi, S. Chen, S.K. Lal, N. Roy Choudhury, S.K. Mukherjee, J. Virol. 78 (2004) 11890–11903. [8] G. Maga, U. Hubscher, J. Cell Sci. 116 (2003) 3051–3060. [9] J.B. Hays, DNA Rep. 1 (2002) 579–600. [10] Y. Uchiyama, S. Kimura, T. Yamamoto, T. Ishibashi, K. Sakaguchi, Eur. J. Biochem. 271 (2004) 2799–2807. [11] S. Roy, S.N. Sarkar, S.K. Singh, D.N. Sengupta, FEBS J. 274 (2007) 2005–2023. [12] S. Das, A. Pal, J. Plant Biochem. Biotechnol. 12 (2003) 11–18. [13] S. Gupta, M.K. Chattopadhyay, P. Chatterjee, B. Ghosh, D.N. SenGupta, Plant Mol. Biol. 37 (1998) 629–637. [14] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254. [15] J. Sambrook, E.F. Fritsch, T. Maniatis, Molecular cloning: A Laboratory ManualIn Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, 1989. [16] S.N. Sarkar, S. Bakshi, M. Sanathkumar, S. Roy, D.N. Sengupta, Biochem. Biophys. Res. Commun. 320 (2004) 145–155. [17] M. Sanathkumar, B. Ghosh, D.N. Sengupta, Biochem. Mol. Biol. Int. 39 (1996) 117–136. [18] S.H. Lee, A.D. Kwong, Z.Q. Pan, J. Hurwitz, J. Biol. Chem. 266 (1991) 594–602.