Nagma Khan
email: nagmabkhan@yahoo.co.uk

Michael Jeffers

Sampath Kumar

Craig Hackett

Ferenc Boldog

Nicholai Khramtsov

Xiaozhong Qian

Evan Mills

Stanny C Berghs

Nessa Carey

Paul W Finn

Laura S Collins

Anthony Tumber

Jamesw Ritchie

Peter Buhl Jensen

Henri S Lichenstein

Maxwell Sehested

Determination of the class and isoform selectivity of small molecule HDAC inhibitors

Keywords: HDAC, HDAC isoform, inhibitors, deacetylation, tubulin HDACs, Histone deacetylases, SMRT, silencing mediator for retinoid and thyroid receptors, NCoR, nuclear receptor corepressor, DAD, deacetylase activating

The human histone deacetylase (HDAC) family, a well-validated anti-cancer target, plays a key role in the control of gene expression through regulation of transcription.

While HDACs can be subdivided into three main classes, the class I, class II and class III HDACs (Sirtuins), it is presently unclear whether inhibiting multiple HDACs using pan HDAC inhibitors, or targeting specific isoforms that show aberrant levels in tumors, will prove more effective as an anti-cancer strategy in the clinic.

To address the above issues, we have tested a number of clinically relevant HDAC inhibitors (HDACi) against a panel of recombinant human HDAC (rhHDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system, and a Fluor de Lys™ (FDL) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDAC inhibitors on class I isoforms (rhHDAC1, 2, 3 and 8) and class II HDAC isoforms (rhHDAC 4, 6, 7 and 9) was determined. MS-275 was HDAC1 selective, MGCD0103 was HDAC1 and HDAC2 selective, apicidin was HDAC2 and HDAC3 selective and valproic acid was an HDAC class I specific

Introduction

Histone deacetylases (HDACs) are a family of enzymes involved in the regulation of a number of cellular processes [START_REF] De Ruijter | Histone deacetylases (HDACs): characterization of the classical HDAC family[END_REF], including cell proliferation, apoptosis and assembly of the cytoskeleton. However, the best characterized HDAC function is the control of gene expression through regulation of transcription [START_REF] Cheung | Acetylation and chromosomal functions[END_REF][START_REF] Cress | Histone deacetylases, transcriptional control, and cancer[END_REF]. This involves an interplay between histone acetyltransferases (HATs) and HDACs, both of which are involved in post-translational modification of histone proteins [START_REF] Strahl | The language of covalent histone modifications[END_REF]. HATs and HDACs have opposing roles in acetylating and deacetylating highly conserved lysine residues on the N-terminal tail of histones, thus altering chromatin assembly and transcriptional activity [START_REF] Wade | Transcriptional control at regulatory checkpoints by histone deacetylases: molecular connections between cancer and chromatin[END_REF][START_REF] Thiel | How mammalian transcriptional repressors work[END_REF]. HDACs are also involved in regulating the acetylation of a number of non-histone proteins, such as α-tubulin and p53 [START_REF] Matsuyama | In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation[END_REF][START_REF] Glaser | Differential protein acetylation induced by novel histone deacetylase inhibitors[END_REF][START_REF] Marks | Histone deacetylases and cancer: causes and therapies[END_REF]. These data, and reports of aberrant HDAC activity in a number of tumor types [START_REF] Glozak | Acetylation and deacetylation of non-histone proteins[END_REF], suggest that inhibition of HDACs represents a viable anti-cancer strategy [START_REF] Johnstone | Histone-deacetylase inhibitors: novel drugs for the treatment of cancer[END_REF][START_REF] Yoshida | Histone deacetylase as a new target for cancer chemotherapy[END_REF][START_REF] Yoshida | Protein deacetylases: enzymes with functional diversity as novel therapeutic targets[END_REF]. functional similarities. The class I isoforms (HDACs 1, 2, 3 and 8) and class II (HDACs 4, 5, 6, 7, 9, 10 and 11) are Zn-dependent enzymes, whereas class III HDACs (Sirtuins (Sir1-7)), are NAD-dependent [START_REF] Bertos | Class II histone deacetylases: structure, function, and regulation[END_REF][START_REF] Gregoretti | Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis[END_REF]. HDACs exist in large multiprotein complexes, and there is evidence that most, if not all, HDAC isoforms require interaction with other HDACs or proteins for optimal enzymatic activity. For example, HDAC3 activity requires an interaction with SMRT/NCoR [START_REF] Guenther | The SMRT and N-CoR corepressors are activating cofactors for histone deacetylase 3[END_REF][START_REF] Codina | Enzymatic activity associated with class II HDACs is dependent on a multiprotein complex containing HDAC3 and SMRT/N-CoR[END_REF], whereas suppression of HDAC4 binding by the HDAC3 -SMRT/NCoR complex results in the loss of HDAC4 enzymatic activity [18]. More recently, the presence of two intramolecular or intermolecular catalytic domains was reported to be required for protein deacetylation [START_REF] Zhang | Two catalytic domains are required for protein deacetylation[END_REF].

A number of HDAC inhibitors are currently undergoing clinical trials as anti-cancer drugs [START_REF] Minucci | Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer[END_REF][START_REF] Yoo | Epigenetic therapy of cancer: past, present and future[END_REF][START_REF] Mclaughlin | Histone deacetylase inhibitors open new doors in cancer therapy[END_REF], some of which have been characterized as selective HDAC inhibitors, and some as pan HDAC inhibitors. Recently vorinostat was registered as an HDACi drug (Zolinza). In the present study, we provide a comprehensive evaluation of the potency and selectivity of various HDAC inhibitors versus recombinant HDAC isoforms.

Materials and Methods

Belinostat, MS-275, vorinostat, NVP-LAQ824, panobinostat, ITF2357 and MGCD0103 were synthesized as described in recent patent applications (International publication number -belinostat: US6888027, MS-275: US6174905, vorinostat: US5369108, ITF2357: WO03/013493, NVP-LAQ824: US6552065, panobinostat: US6552065 and MGCD0103: US6897220). All solvents were purified before use by routine techniques. The reaction products were isolated by evaporating the solvent (vacuum rotary evaporator). Compounds were purified using column chromatography on a silicagel, 0.035-0.070 mm column (Acros). Reversed phase HPLC on a Varian ProStar HPLC system, equipped with a spectrophotometer; was used to check purity of the synthesized compounds. The melting point was determined with micro melting point apparatus (Boëtius or Fisher) and the uncorrected values were used. NMR spectra, obtained on Varian WH-90/DS or Mercury 200 spectrometers, were used to The Bac-N-Blue™ Baculovirus System was used to produce recombinant HDAC isoforms with no fusion protein; however Flag tags were added to the C-termini. In this system the cDNAs were first subcloned into the transfer vector pBlueBac4.5. Sf9 cells grown in adherent format were co-transfected with the transfer vector carrying the HDAC coding sequence and Bac-N-Blue™ linearized baculovirus DNA.

Recombinant viruses were collected and cloned in plaque assays. Six to ten virus clones were tested in a small scale infection for their ability to produce recombinant HDAC and to generate low titer virus stock. One of these virus stocks was selected for generating high-titer virus stock and large-scale infection. High-titer virus stocks were produced by infecting a 200-ml culture of Sf9 cells (1.5 -2.0 X 10 6 cells/ml) with 200 μl of the low-titer stock. Virus-laden media were harvested when the cell viabilities dropped to 40 % or below (4-6 days post infection). Titer of the viral stocks was determined using the FastPlax™ Titer Kit (Novagen, San Diego, CA).

HDAC isoforms with N-terminal fusion of Glutathione-S-Transferase (GST) with or without Flag tags were produced using the Bac-to-Bac® Baculovirus Expression System. The transfer vector pFastBac1 was first modified to include the GST coding sequence at the 5' end of the multiple cloning site. The HDAC cDNAs were inserted downstream of the GST coding sequence through subcloning. Competent E. coli DH10Bac cells were transformed with the recombinant transfer vector. Recombinant bacmids were isolated and verified for the presence of the cDNAs coding for HDACs by PCR, using appropriate primers. Sf9 cells were transfected with the bacmid, and the resulting recombinant viruses were tested for their ability to produce recombinant HDACs. This low-titer stock was used to generate a larger scale and high-titer viral stock as described above.

Large-scale infections

Production cultures were performed either in two to four 3-L shake flasks (1 L culture volume per flask) or in 20-L wave bioreactors (10 L culture volume, Somerset, NJ). appropriate recombinant baculovirus stocks at multiplicities of infection of 3 to 5. Three days post infection; cells were harvested by centrifugation at 1200 X g for 15 min at 4 °C in a Sorvall Evolution RC centrifuge (New Town, CT). Cell pellets were stored at -80 °C until protein purification.

Purification of recombinant HDAC isoforms

Lysis of infected cell pellets

Infected cells were resuspended in ice-cold lysis buffer (50 mM sodium phosphate, 300 mM NaCl, 1 % Triton-X-100, pH 8.0), using 1 ml of lysis buffer for every 2 X 10 7 cells. Complete EDTA-free protease inhibitor cocktail tablets (Roche Applied Science, Indianapolis IN) were dissolved in the lysis buffer just prior to use at a ratio of one tablet for every 50 ml of buffer. The suspension was homogenized for 1 min at a setting of 1 on a T 25 basic ULTRA-TURRAX dispersing instrument (IKA Works, Wilmington NC) and incubated on ice for 45 minutes. Insoluble material was removed by centrifugation at 23,000 X g at 4 ºC for 45 minutes in a Sorvall Evolution RC centrifuge (Newtown CT). The clarified lysate was processed immediately by batch chromatography.

Batch-mode affinity purification using glutathione sepharose

GST-tagged proteins were purified using Glutathione Sepharose 4 Fast Flow (GE, Piscataway NJ) (Supplementary Table 2). Settled Sepharose beads were washed by suspending in a 10-fold excess of ice-cold lysis buffer followed by centrifugation at 4 ºC for 5 minutes at 213 X g in a Sorvall Legend RT tabletop centrifuge equipped with a swinging-bucket rotor. The wash step was repeated two more times. The washed beads were suspended in lysis buffer to make a 50 % slurry. The lysate was split into 50-ml aliquots in disposable centrifuge tubes, and 1.25 ml of Sepharose slurry was added to each tube. Tubes were incubated for one hour at 4 ºC with end-over-end mixing. The charged matrix was collected by centrifugation, and the beads were washed by centrifugation three times with 40 ml of ice-cold PBS. Finally, the matrix was resuspended in PBS and transferred to a Poly-Prep disposable chromatography column (Bio-Rad Laboratories, Hercules CA). The column was placed at 4 ºC and Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. allowed to drain until the top of the bed was dry. One-third packed bed volume of icecold elution buffer (50 mM HEPES, 40 mM reduced L-glutathione, pH 9.0) was carefully added to the top of the bed and allowed to drain through the column. The effluent was discarded. Two packed bed volumes of elution buffer were carefully added to the top of the bed, and the effluent was collected as a single fraction. The effluent containing the HDAC was immediately aliquoted and frozen at -80 ºC.

Batch-mode affinity purification using anti-FLAG agarose

FLAG-tagged proteins were purified using ANTI-FLAG M2 Affinity Gel (Sigma, St.

Louis MO) (Supplementary Table 2). Settled agarose beads were washed as described above, twice with ice-cold PBS, once with 0.1 M glycine, pH 2.8, and twice with PBS containing an additional 150 mM NaCl. The washed beads were suspended in PBS containing an additional 150 mM NaCl to make a 50 % slurry. Batch-mode capture, washing with PBS, and transfer to a Poly-Prep chromatography column were performed as described above. The column was placed at 4 ºC and allowed to drain until the top of the bed was dry. Elution buffer was prepared as follows. 3X FLAG peptide (Sigma, St. Louis MO) was dissolved at a concentration of 25 µg/ml in 0.5 M Tris-HCl, 1 M NaCl, pH 7.5 and the solution was diluted 1:4 with water to make a 5 µg/ml stock which was stored at -20 ºC. Elution buffer was prepared just prior to use by adding the 3 X FLAG peptide stock to PBS to a final concentration of 250 ng/ml. The pre-elution and elution steps, and sample aliquoting were performed as described above.

HDAC assays

Deacetylase activity of rhHDACs1, 2, 3, 4, 6, 7 and 9 was assayed with a pan HDAC substrate (initially KI-104, Biomol and subsequently I-1925, Bachem). Deacetylase activity of rhHDAC8 was assayed with HDAC8 specific Fluor de Lys™ (FDL) substrate (KI-178, Biomol). The total pan HDAC assay volume was 50 µl and all the assay components were diluted in Hepes buffer (25 mM Hepes, 137 mM NaCl, 2.7 mM KCl, 4.9 mM MgCl 2 , pH. 8.0). The reaction was carried out in half volume white 96-well plates (Costar 3693). In brief, the pan HDAC assay contained HDAC Positive controls contained all the above components, except the inhibitor. The negative controls did not contain either enzyme or inhibitor. In each case these were replaced with an equivalent volume of buffer. The assay components were incubated at 37°C for 3 h. The reaction was quenched with the addition of 50 µl HDAC-FDL Developer (KI-105, Biomol) 20 X stock diluted to 1:400 in Hepes buffer and containing 2 µM TSA. The plates were incubated (25 min) at room temperature to allow the fluorescent signal to develop. The fluorescence generated was monitored at 355/460 nm (excitation/emission) wavelength.

The HDAC8 specific assay was as described for the pan HDAC assay, except for the changes indicated. The Hepes assay buffer contained BSA (1 mg/ml). The FDL-HDAC8 substrate was 25 µM. The assay components were incubated (3 h) at room temperature and then the reaction was quenched with HDAC-FDL Developer (KI-176, Biomol) 5 X stock diluted 1:110 in Hepes buffer containing BSA (1mg/ml) and 2 µM TSA. The plates were incubated (25 min) at 4°C to allow the fluorescent signal to develop.

Cell proliferation assays

HeLa cells were grown in monoculture and treated with serial dilutions of the HDACi. The cells were incubated (48 h) at 37°C. The number of viable cells was assessed using Cell Proliferation Reagent WST-1. Media only and HeLa cells without HDACi were used as negative and positive controls, respectively.

Immunoblotting

Cells grown in monolayer culture were treated with compounds as indicated in the figure legends. Cells were harvested in 2 X Tris-glycine SDS sample buffer (Invitrogen) supplemented with 10 mM DTT (Sigma). Cell lysates were boiled for 10 minutes, resolved on 4-20% gradient SDS-polyacrylamide gels (Invitrogen), and transferred to nitrocellulose membranes (Invitrogen). Immunoblotting was performed using standard procedures and the following primary antibodies: anti-acetylated Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

(lys40) alpha-tubulin (Sigma, T6793), anti-acetylated (lys9) histone H3 (Cell Signaling Technology, 9671), anti-acetylated lysine (Ac-K-103, Cell Signaling Technology, 9681), anti-p53 (mix of antibodies from Santa Cruz Biotechnology, sc-126 and Calbiochem, OP03) and anti-actin (Sigma, A5060). Following incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies, enhanced chemiluminescence (GE Healthcare) was used for detection.

Results

Cloning and Expression

Eight full length recombinant human HDACs (rhHDAC) were expressed in insect cells using a baculoviral expression system. The purified rhHDACs consisted of four class I enzymes (FLAG-HDAC1, GST-HDAC2-FLAG, FLAG-HDAC3+FLAG-SMRT and GST-HDAC8) and four class II enzymes (GST-HDAC4-His, His-HDAC6, HDAC7-FLAG and GST-HDAC9-FLAG.

HDAC assay

Various hydroxamate and non-hydroxamate based HDAC inhibitors (Table 1), were tested against purified rhHDAC proteins using a FDL assay and EC 50 values were determined (Table 2). Of the ten inhibitors included in this study, the hydroxamic acid derivatives proved to be the most potent HDAC inhibitors, showing activity on both class I and class II HDAC isoforms. The most potent compounds, TSA, NVP-LAQ824 and panobinostat, exhibited EC 50 values in the low nM range against rhHDACs 1, 2, 3, 4 and 9, and were 5-30 fold more potent than vorinostat and belinostat. TSA was also >10 fold more potent than vorinostat and belinostat as an inhibitor of rhHDAC 6 and 7, and was approximately equipotent to belinostat as an inhibitor of rhHDAC 8. NVP-LAQ824 and panobinostat were somewhat more potent as inhibitors of rhHDAC 6 and 7 compared to vorinostat and belinostat, and were equipotent as inhibitors of rhHDAC 8 compared to belinostat. Belinostat was approximately equipotent as an inhibitor of all rhHDAC isoforms tested. Vorinostat exhibited a similar profile, although it appeared to be slightly less effective as an In order to make a clear comparison of the potency of the inhibitors on the rhHDAC, log EC 50 values were plotted for all compounds with reference to the 2 major HDAC classes (Figure 1a and1b). Of the class I HDACs, rhHDAC8 was an outlier, as most compounds tested were less potent against this isoform than against rhHDACs 1, 2 and 3 (Figure 1a). MS-275, MGCD0103, apicidin, valproic acid had little activity on class II isoforms, thus confirming that these compounds are class I selective (Figure 1b).

Cell proliferation assay

Ten hydroxamate and non-hydroxamate HDAC inhibitors (Table 3) were tested in cell proliferation assays in HeLa cells. NVP-LAQ824, panobinostat, TSA and ITF-2357 exhibited the highest potency with EC 50 s <1 µM. MGCD0103, apicidin and belinostat had EC 50 s <2 µM. Vorinostat and MS-275 had an EC 50 of 3.2 µM and 3.9 µM, respectively. VPA had an EC 50 of 7.3 mM. Belinostat was found to be 1.7 fold more potent than vorinostat in the HeLa cell proliferation assay.

Determination of acetylation of levels of proteins after HDACi treatment

The lysates of HeLa cells exposed to HDAC inhibitors at 2 X EC 50 , for 4 h and 24 h, were examined by immunoblotting with anti-acetyl-lysine antibody. Both the pan HDAC and the isoform-specific inhibitors induced the acetylation of low molecular weight proteins consistent with the size of histones H3 and H4 (17-kDa and 11-kDa, respectively) in the lysates of cells treated with inhibitor for 24 h. However, the lysates from cells exposed to MS-275 and MGCD0103 for 4 h showed a lower level of acetylation of the two low molecular weight proteins (Figure 2a), compared with lysates from cells treated with the same compounds for 24 h (Figure 2b). In addition, the pan HDAC inhibitors induced the acetylation of a high molecular weight protein, whose size is consistent with that of tubulin (Figure 2a and2b).

HeLa cells were exposed to the HDACi at 8 X their EC 50 , for 24 h, and the whole cell lysates were examined by immunoblotting for acetyl tubulin and acetyl histone H3.

The results (Figure 3) show that, compared to the control lysate, the pan HDAC inhibitors (belinostat, vorinostat, TSA, NVP-LAQ824, panobinostat and ITF2357) induced strong acetylation of tubulin and histone H3, while the isoform-specific inhibitors induced strong acetylation of histone H3 but weak or no acetylation of tubulin.

HCC827 and DU145 cells were treated for 24 h with belinostat, MS-275 and MGCD0103 at four different concentrations (0.125 X, 0.25 X, 0.5 X and 1 X EC 50 ).

The lysates from these cells were examined by immunoblotting with an anti-p53 antibody. The results for both cell lines showed that belinostat decreased the expression of p53 in a dose dependent manner, while the HDAC isoform-specific compounds MS-275 and MGCD0103 did so only weakly, if at all (Figure 4a andb).

DISCUSSION

Recently published data indicate that both class I and class II HDACs are aberrantly expressed in human cancers. HDAC1 is up-regulated in prostate cancer [START_REF] Halkidou | Upregulation and nuclear recruitment of HDAC1 in hormone refractory prostate cancer[END_REF] and gastric cancer [START_REF] Choi | Expression profile of histone deacetylase 1 in gastric cancer tissues[END_REF], HDAC2 is up-regulated in gastric cancer [26], HDAC3 is upregulated in lung cancer [START_REF] Bartling | Comparative application of antibody and gene array for expression profiling in human squamous cell lung carcinoma[END_REF], and there is elevated expression of HDAC6 in oral squamous cell carcinoma [START_REF] Sakuma | Aberrant expression of histone deacetylase 6 in oral squamous cell carcinoma[END_REF]. HDAC8 has also recently been implicated in contributing to tumorigenesis by regulating telomerase activity, via its interaction with human ever-shorter telomeres 1B (hEST1B) [START_REF] Lee | Histone deacetylase 8 safeguards the human ever-shorter telomeres 1B (hEST1B) protein from ubiquitin-mediated degradation[END_REF].

The main objective of this study was to compare the potency and specificity of a number of structurally diverse small molecule HDACi compounds as inhibitors of class I or II HDAC isoforms. A number of these inhibitors, including MGCD0103, vorinostat, panobinostat, valproic acid, MS-275, ITF2357 and belinostat, are currently undergoing Phase I, Phase II and Phase III clinical trials as anti-cancer drugs. A further aim was to determine the mode of action of these inhibitors, in particular their effect on the induction of acetylation of specific proteins in cells.

Purified class I and class II HDACs were obtained to address isoform specificity. Four class I and four class II active rhHDAC isoforms were expressed and purified, and although co-purification of a small number of endogenous proteins was noted in the preparations, these were always minor components compared to the recombinant enzyme. The exception was rhHDAC3 preparations, which were all inactive without the presence of SMRT protein. As a result of this requirement, rhHDAC3 was coexpressed with SMRT protein resulting in a highly active enzyme.

Our findings suggest that hydroxamic acid derived compounds such as TSA, NVP-LAQ824, panobinostat, ITF2357, vorinostat and belinostat act as potent pan HDAC isoform inhibitors. A notable observation was the similarity between belinostat and vorinostat in the biochemical isoform assays; both compounds exhibit similar EC 50 values in all but the HDAC8 assay. Although vorinostat and belinostat appear similar in biochemical assays, belinostat is ~ 1.7 -7 fold more potent as an inhibitor of cell proliferation in a number of cancer cell lines (Table 3; unpublished data from TopoTarget and CuraGen; NCI data (http://dtp.nci.nih.gov; Belinostat (NSC# 726630), Vorinostat (NSC# 701852)).

NVP-LAQ824 was more potent than TSA or panobinostat in the HeLa growthinhibition assay (Table 3). The reason for this is unknown, and is somewhat surprising given the similarity in the enhanced induction of H3/H4 acetylation (Figures 2a, 2b 2). However, it should be pointed out that the biochemical assay is performed in a cell-free system whereas the growth-inhibition assay is performed on intact cells. Potential reasons for the observed enhanced growthinhibitory activity of NVP-LAQ824, relative to TSA or panobinostat, include: [START_REF] De Ruijter | Histone deacetylases (HDACs): characterization of the classical HDAC family[END_REF] greater cellular uptake or retention of NVP-LAQ824; (2) superior access of NVP-LAQ824 to intracellular targets following uptake; (3) longer half-life of NVP-LAQ824 in intact cells; (4) greater off-target effects of NVP-LAQ824.

In contrast to the hydroxamates, the benzamide compounds MS-275 and MGCD0103, appeared to be HDAC class I specific, as reported previously [START_REF] Hu | Identification of novel isoformselective inhibitors within class I histone deacetylases[END_REF][START_REF] Eyupoglu | Experimental therapy of malignant gliomas using the inhibitor of histone deacetylase MS-275[END_REF][START_REF] Kalita | Pharmacodynamic effect of MGCD0103, an oral isotype-selective histone deacetylase (HDAC) inhibitor, on HDAC enzyme inhibition and histone acetylation induction in phase I clinical trials in patients (pts) with advanced solid tumors or non-Hodgkin's lymphoma (NHL)[END_REF][START_REF] Gelmon | Phase I trials of the oral histone deacetylase (HDAC) inhibitor MGCD0103 given either daily or 3x weekly for 14 days every 3 weeks in patients (pts) with advanced solid tumors[END_REF]. Our findings for MS-275 inhibition of HDACs 1, 3 and 8 agree with published data [START_REF] Hu | Identification of novel isoformselective inhibitors within class I histone deacetylases[END_REF].

The small chain fatty acid compound, valproic acid has been shown to inhibit HDACs [START_REF] Gurvich | Association of valproate-induced teratogenesis with histone deacetylase inhibition in vivo[END_REF] and to selectively induce proteosomal degradation of HDAC2 [START_REF] Kramer | The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2[END_REF]. VPA has been used, both as monotherapy and in combination with a number of drugs, in the treatment of cancer [START_REF] Kaiser | The effects of the histone deacetylase inhibitor valproic acid on cell cycle, growth suppression and apoptosis in multiple myeloma[END_REF][START_REF] Kuendgen | The histone deacetylase (HDAC) inhibitor valproic acid as monotherapy or in combination with all-trans retinoic acid in patients with acute myeloid leukemia[END_REF][START_REF] Morotti | Valproate enhances imatinib-induced growth arrest and apoptosis in chronic myeloid leukemia cells[END_REF]. Our data indicates that VPA inhibited class I HDACs 1, 2, 3 and 8 in the millimolar range, but it was ineffective against HDACs 6, 7 and 9.

When comparing the class I HDACs, the EC 50 values for rhHDAC8 were consistently higher for all HDAC inhibitors tested, compared with the other rhHDAC isoforms. This phenomenon may be explained by recent data on HDAC8, which shows that HDAC8 affinity for vorinostat is influenced by the identity of the divalent ion present in the catalytic centre. The Km values with Fe(II) and Co(II) were shown to be 5-fold lower than with Zn(II). This data indicates that HDAC8 requires Fe(II) as the divalent ion for catalytic activity, rather than Zn(II) [START_REF] Gantt | Catalytic activity and inhibition of human histone deacetylase 8 is dependent on the identity of the active site metal ion[END_REF]. This difference was not apparent after 24 h inhibitor treatment. A possible explanation for this finding is that MS-275 and MGCD0103 may have slower kinetics than the other drugs. These findings indicate that both pan HDAC and class I specific inhibitors bring about similar changes to the level of acetylation of lysine residues on histones H3 and H4.

Interestingly, class II HDACs, particularly HDAC6, appear to be important in a number of key cellular processes. Our data showed that treatment of HeLa cells with HDACi, followed by immunoblotting of the cell extracts with anti-acetylated tubulin antibody resulted in increased tubulin acetylation in extracts treated with the pan HDAC inhibitors; this was not observed with the class I specific inhibitors. There is evidence that HDAC6 destabilizes the microtubule assembly by deacetylating tubulin via its tubulin deacetylase domain [START_REF] Matsuyama | In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation[END_REF][START_REF] Hubbert | HDAC6 is a microtubuleassociated deacetylase[END_REF][START_REF] Zhang | HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo[END_REF]. HDAC6 also plays a role in aggresome function [START_REF] Kawaguchi | The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress[END_REF]. Furthermore, there is evidence that inhibition of HDAC6 results in increased acetylation of HSP90 and disruption of the chaperone association with its client proteins [START_REF] Bali | Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90: a novel basis for antileukemia activity of histone deacetylase inhibitors[END_REF]. p53 is a negative regulator of cell growth. It is expressed in the mutant form in a high proportion of tumor cell types. Mutant p53 is known to be a "client" protein of HSP90 [START_REF] Sharp | Inhibitors of the HSP90 molecular chaperone: current status[END_REF].

In order to determine if there were any marked differences in p53 levels in cells treated with pan HDAC and class I specific inhibitors, the lysates from DU145 cells and HCC827 cells were immunoblotted with an anti-p53 antibody, following 24 h treatment with HDAC inhibitors. DU145 cells are known to express mutant p53 and HCC827 cells are likely to express p53. Treatment of cells with belinostat showed a dose dependent decrease in p53 levels in the lysates of both cell types. This depletion of mutant p53 was not observed in the lysates from cells treated with the class I inhibitors MS-275 and MGCD0103. These findings collectively emphasize the importance of HDAC6 and suggest that it could also be an important anti-cancer target. If this hypothesis proves to be correct, a pan HDAC inhibitor may be more effective in the clinic than a class I selective inhibitor.

One theoretical consideration is whether inhibition of specific HDACs by selective inhibitors will prove to be less toxic in the clinic than pan-inhibition of both class I Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Phase II

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Sep 2007 as manuscript BJ20070779 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070779

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Sep 2007 as manuscript BJ20070779 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070779Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  substrate (30 µM, 25 µl); rhHDAC isoform (0.5 -3 µl, diluted to 15 µl final volume) and inhibitor (5 X the required assay concentration, diluted to 10 µl final volume).

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Sep 2007 as manuscript BJ20070779 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070779

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Sep 2007 as manuscript BJ20070779 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070779Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  inhibitor of rhHDAC8. ITF2357 proved to be equipotent to vorinostat and belinostat in the rhHDAC1, 2, 3, 4, 6, and 7 assays. MS-275 exhibited EC 50 values in the nM range against rhHDAC 1. It was significantly less potent (EC 50 values in the low µM range) against rhHDACs 2, 3 and 9. MS-275 did not inhibit rhHDACs 4, 6 7 and 8. Similarly, MGCD0103 exhibited EC 50 values in the low nM range against rhHDACs 1 and 2. However, it was much less potent against rhHDAC3, with an EC 50 value of ~1 µM, and did not inhibit the activity of rhHDAC 8 or class II HDACs. Apicidin inhibited rhHDACs 2 and 3 in the nM range. It also inhibited rhHDAC 8 but with lower potency (EC 50 of 575 nM), and had no inhibitory effect on rhHDAC1 and class II rhHDACs. Valproic acid inhibited rhHDACs 1, 2, 3 and 8 in the mM range, but exhibited little activity in the class II rhHDAC assays; hence potency was several orders of magnitude lower compared with the other compounds tested. Those compounds which gave incomplete inhibition of rhHDAC isoforms at 100 µM were given a value of >100000.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Sep 2007 as manuscript BJ20070779 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070779 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

and 3 )

 3 , and the relative equality of these three compounds in biochemical assays of S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Sep 2007 as manuscript BJ20070779 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070779 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society HDAC inhibition (Table

  To determine possible modes of action of the inhibitors, the change in lysine acetylation of key proteins in HeLa cells was established. Inhibition of the HDACs should result in increased acetylation of specific (ε-N-acetyl lysine) residues on histones. To confirm this hypothesis, HeLa cells were treated with the HDAC inhibitors for 4 h and 24 h; controls were treated with DMSO only. The cell extracts were immunoblotted with anti-acetyl-lysine antibody. Compared with the control extracts, the inhibitor-treated cell extracts showed increased acetylation of two low molecular weight proteins, which corresponded in size to histones H3 and H4. Cells S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Sep 2007 as manuscript BJ20070779 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070779 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society which had been treated with MS-275 and MGCD0103 for 4 h showed a significantly lower level of acetylation of these proteins compared to pan inhibitor-treated cells.

  S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Sep 2007 as manuscript BJ20070779 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070779Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  and II HDACs. Evidence available from early phase clinical trials suggests that toxicities associated with pan HDAC inhibitors, such as belinostat and vorinostat[START_REF] Steele | A phase 1 pharmacokinetic (PK) and pharmacodynamic (PD) study of the histone deacetylase (HDAC) inhibitor PXD101 in patients (pts) with advanced solid tumours[END_REF][START_REF] Duvic | Vorinostat: a new oral histone deacetylase inhibitor approved for cutaneous T-cell lymphoma[END_REF] 47], are no more severe than those observed in patients treated with class I selective inhibitors, such as MS-275 and MGCD0103[48, 49, 50]. The most viable conclusion may therefore be, that until further evidence highlighting the benefits of class I selective inhibitors becomes available, pan HDACi may be advantageous since they inhibit important class II HDAC isoforms such as HDAC 6, without demonstrating increased toxicity. However, the ultimate factor(s) deciding between a pan HDAC or class selective inhibitor may depend on the cancer type and/or the chemotherapeutic combination being tested. S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Sep 2007 as manuscript BJ20070779 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070779

Table 3 .

 3 Each compound was assayed in triplicate per plate. EC 50 values were determined from the average of a minimum of 2 plates. The average EC 50 values and standard error from the mean are shown in nanomolar units S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Sep 2007 as manuscript BJ20070779 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070779 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society 7Inhibition of HeLa cell proliferation by HDAC inhibitors. Each compound was assayed in triplicate per plate. EC 50 values were determined from the average of a minimum of 2 plates. The average EC 50 values and standard error from the mean are shown in micromolar units S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Sep 2007 as manuscript BJ20070779 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070779

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. The inhibitory activity of the indicated HDACi on recombinant class I [1, 2, 3, 8; (a)] and class II [4, 6, 7, 9; (b)] HDAC isoforms was determined in a cell-free system using the Fluor de Lys™ assay and plotted as Log EC 50 for purposes of comparison

Figure 4 .

 4 Figure 4. HCC827 and DU145 cells were exposed to the indicated HDACi at concentrations of 0.125-1 X EC50 (as determined in a cell viability assay on HeLa cells) for 24 h, after which time total cell lysates were prepared and examined by immunoblotting under reducing conditions with an antibody to p53. The blots were reprobed with actin to confirm equal loading S t a g e 2 ( a ) P O S T -P R I N T

  Figure 2a

Table 1 .

 1 HDAC inhibitors S t a g e 2 ( a ) P O S T -P R I N T Biochemical Journal Immediate Publication. Published on 14 Sep 2007 as manuscript BJ20070779 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20070779 Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2007 The Authors Journal compilation © 2007 Biochemical Society

	Inhibitor	rhHDAC1	rhHDAC2	rhHDAC3	rhHDAC4	rhHDAC6	rhHDAC7	rhHDAC8	rhHDAC9
	TSA	2 ± 0	3 ± 0	4 ± 1	6 ± 2	3 ± 1	5 ± 2	456 ± 59	6 ± 5
	NVP-LAQ824	5 ± 1	5 ± 1	12 ± 1	23 ± 18	7 ± 3	18 ± 4	162 ± 44	6 ± 5
	Panobinostat	3 ± 0	3 ± 0	4 ± 1	12 ± 5	61 ± 1	14 ± 7	248 ± 11	3 ± 2
	ITF2357	28 ± 8	56 ± 13	21 ± 3	52 ± 5	27 ± 16	163 ± 8	ND	ND
	Belinostat	41 ± 6	125 ± 21	30 ± 0	115 ± 16	82 ± 19	67 ± 22	216 ± 43	128 ± 46
	Vorinostat	68 ± 14	164 ± 45	48 ± 17	101 ± 31	90 ± 26	104 ± 35	1524 ± 463	107 ± 21
	MS-275	181 ± 62	1155 ± 134	2311 ± 803	>10,000	>10,000	>10,000	>10,000	505 ± 37
	MGCD0103	34 ± 17	34 ± 8	998 ± 431	>10,000	>10,000	>10,000	>10,000	ND
	Apicidin	>10,000	120 ± 28	43 ± 7	>10,000	>10,000	>10,000	575 ± 111	>10,000
	Valproic acid	1,584,000 ± 302,642	3,068,000 ± 0	3,071,000 ± 0	ND	>10,000	>10,000	7,442,000 ± 2,740,000	>10,000

EC 50 ± SEM (nM)

	Inhibitor	rhHDAC1	rhHDAC2	rhHDAC3	rhHDAC4	rhHDAC6	rhHDAC7	rhHDAC8	rhHDAC9
	TSA	2 ± 0	3 ± 0	4 ± 1	6 ± 2	3 ± 1	5 ± 2	456 ± 59	6 ± 5
	NVP-LAQ824	5 ± 1	5 ± 1	12 ± 1	23 ± 18	7 ± 3	18 ± 4	162 ± 44	6 ± 5
	Panobinostat	3 ± 0	3 ± 0	4 ± 1	12 ± 5	61 ± 1	14 ± 7	248 ± 11	3 ± 2
	ITF2357	28 ± 8	56 ± 13	21 ± 3	52 ± 5	27 ± 16	163 ± 8	ND	ND
	Belinostat	41 ± 6	125 ± 21	30 ± 0	115 ± 16	82 ± 19	67 ± 22	216 ± 43	128 ± 46
	Vorinostat	68 ± 14	164 ± 45	48 ± 17	101 ± 31	90 ± 26	104 ± 35	1524 ± 463	107 ± 21
	MS-275	181 ± 62	1155 ± 134	2311 ± 803	>10,000	>10,000	>10,000	>10,000	505 ± 37
	MGCD0103	34 ± 17	34 ± 8	998 ± 431	>10,000	>10,000	>10,000	>10,000	ND
	Apicidin	>10,000	120 ± 28	43 ± 7	>10,000	>10,000	>10,000	575 ± 111	>10,000
	Valproic acid	1,584,000 ± 302,642	3,068,000 ± 0	3,071,000 ± 0	ND	>10,000	>10,000	7,442,000 ± 2,740,000	>10,000

EC 50 ± SEM (nM)

Table 2 .

 2 Inhibition of rhHDAC isoforms in the HDAC in vitro biochemical assay.

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2007 The Authors Journal compilation © 2007 Biochemical Society

Figure 4a


Poonam Taneja

Heinz-Peter Nasheuer

Hella Hartmann

Frank Grosse

Ellen Fanning

Klaus Weißhart
email: weisshart@zeiss.de

Timed interactions between viral and cellular replication factors during the initiation of SV40 in vitro DNA replication

Keywords: SV40 DNA replication, large T-antigen, Replication protein A, DNA polymerase α-primase Simian virus 40, Tag, large T-antigen, Pol, DNA polymerase αprimase, RPA, Replication protein

The initiation of Simian virus 40 (SV40) DNA replication requires the cooperative interactions between the viral large T-antigen (Tag), replication protein A (RPA) and DNA polymerase α-primase (Pol) on the template DNA. Binding interfaces mapped on these enzymes and expressed as peptides competed with these mutual interactions of the native proteins. Prevention of the genuine interactions was accomplished only prior to the primer synthesis step and blocked the assembly of a productive initiation complex. Once the complex was engaged in the synthesis of an RNA primer and its extension, the interfering effects of the peptides ceased, suggesting a stable association of the replication factors during the initiation phase. Specific antibodies were still able to disrupt preformed interactions and inhibited primer synthesis and extension activities underlining the crucial role of specific protein-protein contacts during the entire initiation process.

INTRODUCTION

Simian virus 40 (SV40) provides an effective model system to study eukaryotic DNA replication [START_REF] Kelly | SV40 DNA replication[END_REF]. One viral protein, large T-antigen (Tag), orchestrates the entire replication of the viral mini-chromosome in primate cellular extracts [START_REF] Stenlund | Initiation of DNA replication: lessons from viral initiator proteins[END_REF]. Tag assembles as a double hexamer on its cognate origin sequences, distorts and bi-directionally unwinds the duplex DNA in an ATP-dependent manner and recruits other cellular replication proteins to assemble the replication fork. For the initiation reaction, three cellular proteins are involved in addition to Tag. Topoisomerase I (Topo) releases torsional stress induced by the helicase activity of Tag ahead and behind of the replication fork. The heterotrimeric replication protein A (RPA) is required to stabilize emerging single stranded DNA (ssDNA) regions and in conjunction with Tag enables DNA polymerase α-primase (Pol) to synthesize and extend primers [START_REF] Simmons | SV40 large T antigen functions in DNA replication and transformation[END_REF]. For subsequent steps the host replication machinery provides all other necessary factors [START_REF] Waga | Reconstitution of complete SV40 DNA replication with purified replication factors[END_REF].

The coordination of the individual steps during the replication reaction relies on the modular design of replication factors and their organization into structural and functional domains [START_REF] Stauffer | Structural mechanisms of DNA replication, repair, and recombination[END_REF].

Specific interactions have been attributed to certain subunits or domains of Tag, RPA, Pol and Topo and the contact sites between these key players are crucial for the initiation process [START_REF] Dornreiter | Interaction of DNA polymerase α-primase with cellular replication protein A and SV40 T antigen[END_REF][START_REF] Kim | Role of the 70-kDa subunit of human replication protein A (I). Single-stranded DNA binding activity, but not polymerase stimulatory activity, is required for DNA replication[END_REF][START_REF] Braun | Role of proteinprotein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase α by multiple mechanisms[END_REF][START_REF] Weisshart | The replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication[END_REF][START_REF] Arunkumar | Insights into hRPA32 C-terminal domain-mediated assembly of the simian virus 40 replisome[END_REF][START_REF] Roy | The cap region of topoisomerase I binds to sites near both ends of simian virus 40 T antigen[END_REF]. Tag amino acids within the origin DNA binding domain (OBD) have been identified to interact with sequences within the DNA binding domain A (DBD-A) of the largest p70 subunit of RPA. The same region of p70 and stretches in its N-terminal domain contact the largest p180subunit of Pol, which in turn binds the N-terminal and helicase domains of Tag via a region situated N-terminal to the catalytic polymerase domain. Some of the activities of the single replication factors correlate well with the physical interactions that they undergo [START_REF] Dornreiter | Interaction of DNA polymerase α-primase with cellular replication protein A and SV40 T antigen[END_REF][START_REF] Roy | The cap region of topoisomerase I binds to sites near both ends of simian virus 40 T antigen[END_REF][START_REF] Melendy | An interaction between replication protein A and SV40 T antigen appears essential for primosome assembly during SV40 DNA replication[END_REF][START_REF] Collins | The effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase α-primase[END_REF]. The precise timing of these interactions, however, and the steps when they are required, are only starting to emerge [START_REF] Arunkumar | Insights into hRPA32 C-terminal domain-mediated assembly of the simian virus 40 replisome[END_REF][START_REF] Simmons | Assembly of the replication initiation complex on SV40 origin DNA[END_REF].

Here we employ peptides that interfered with specific individual associations between Tag, RPA and Pol. Mutual interactions of these factors became first important during the assembly of a meta-stable pre-initiation complex. The transition to the initiation phase was accompanied by a tightening of these interactions within the initiation complex (primosome).

MATERIALS AND METHODS

Cloning of fusion peptides

Sequences for RPA, Tag and Pol were amplified from cloned genes using primer pairs R70.1N and R70.173C for peptide R1-173; R70.174N and R70.250C for peptide R174-250 (both RPA); T96. 164N

Purification of replication proteins and MBP fusion peptides

SV40 large T-antigen (Tag) and the human DNA polymerase α-primase complex (Pol) were expressed in Sf9 cells infected with recombinant Baculo viruses and purified as described [START_REF] Dornreiter | SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha[END_REF][START_REF] Stadlbauer | DNA replication in vitro by recombinant DNApolymerase-α-primase[END_REF]. His-tagged Topoisomerase I (Topo) expressed in Baculo virus was purified on Talon-resin (Clontech) and kindly provided by Kent Søe [START_REF] Soe | A human topoisomerase I cleavage complex is recognized by an additional human topisomerase I molecule in vitro[END_REF]. Bacterially expressed his-tagged replication protein A (H 6 -RPA) was purified as outlined for RPA [START_REF] Henricksen | Recombinant replication protein A: expression, complex formation, and functional characterization[END_REF]. MBP-fusion peptides were expressed and purified on amylose resin as described [START_REF] Kolpashchikov | Interaction of the p70 subunit of RPA with a DNA template directs p32 to the 3'-end of nascent DNA[END_REF].

Protein-protein interactions

For co-precipitation assays [START_REF] Weisshart | Protein-protein interactions of the primase subunits p58 and p48 with simian virus 40 T antigen are required for efficient primer synthesis in a cell-free system[END_REF] 50 pmol of specific monoclonal antibodies (0.15 µg/pmol), Pab101 for TAg [START_REF] Gurney | Monoclonal antibodies against simian virus 40 T antigens: evidence for distinct sublcasses of large T antigen and for similarities among nonviral T antigens[END_REF], SJK237-87 for Pol [START_REF] Tanaka | Preparation and preliminary characterization of monoclonal antibodies against human DNA polymerase α[END_REF], and anti his-tag (Qiagen) for H 6 -RPA were coupled to 20 µl 50 % (v/v) protein G-agarose beads in 100 µl binding buffer (50 mM Hepes-KOH 7.9; 100 mM KCl; 7 mM MgCl 2 ; 0.25% Inositol; 0.25 mM EDTA; 0.05 % NP-40) for 1 hr at 4 °C. Alternatively 50 µl of polyclonal anti RPA serum were used. The beads were then washed 4 times with 1 ml wash buffer (30 mM Hepes-KOH pH7.9; 100 mM KCl; 7 mM MgCl 2 ). 25 pmol of Tag (0.096 µg/pmol), Pol (3.54 µg/pmol) or RPA (0.116 µg/pmol) were added to the corresponding beads in a 100 µl reaction in binding buffer. Alternatively, MBP-fusion peptides served as the bait and were coupled to amylose resin at a concentration of 1.25 pmol/µl. After 1 hr at 4 °C to immobilise the bait protein, the beads were washed 4 times with 1 ml wash buffer. The beads were then incubated with 25 pmol of the target protein in the absence or presence of a 25 to 100 molar excess of peptides over the target protein or 50 pmol antibodies in 100 µl reaction buffer containing 2% BSA for 2 h at 4 °C. Alternatively antibodies were added after the target protein was allowed to bind to determine their disruption capability on pre-formed protein-protein interactions. In these cases incubation continued for another hour. After washing 4 times as before, the beads were boiled in 20 µl sample buffer. Eluted proteins were run on a 10% SDS-polyacrylamide gel and detected after western blotting with the enhanced chemiluminescence (ECL) system (GE Healthcare). Quantification of bands was done by densitometry. To this end, lanes of underexposed films (to stay as linear as possible) were profiled and the area under the scan curve was used as an estimation of the co-precipitated protein amount compared to standards run in parallel. A total of five independent experiments were averaged.

Mono-polymerase system

All reactions contained in an end volume of 40 µl 30 mM Hepes-KOH pH 7.8; 7 mM MgAc; 1 mM EGTA; 0.5 mM DTT; 40 mM creatine-phosphate; 40 µg/µl creatinekinase; 4 mM ATP, 0.2 mM of each CTP, GTP and UTP; 0.2 mM each dATP and dGTP ; 0.05 mM each dCTP and dTTP ; 10 µCi each α 32 P-dCTP and α 32 P-dTTP; 1.2 pmol human Topoisomerase I (Topo, 0.1 µg/pmol) ; 3.5 pmol replication protein A (RPA, 0.116 µg/pmol); 6.25 pmol SV40 large T-antigen (Tag, 0.096 µg/pmol); 1.1 pmol human DNA polymerase α-primase (Pol, 0.354 µg/pmol) and 0.1 pmol pUC-HS DNA (2 µg/pmol) containing the complete SV40 origin sequences [START_REF] Weiner | Specific mutation of a regulatory site within the ATP-binding region of simian virus 40 large T antigen[END_REF]. Since Pol will synthesize the leading and lagging strands in this type of reaction the assay is refered to as the mono-polymerase system [START_REF] Murakami | DNA polymerase α stimulates the ATP-dependent binding of simian virus tumor T antigen to the SV40 origin of replication[END_REF]. For SV 40 in vitro DNA replication Topo, RPA and Pol were replaced by 190 µg S100 extracts otained from human 293 cells [START_REF] Guo | Origin auxiliary sequences can facilitate initiation of simian virus 40 DNA replication in vitro as they do in vivo[END_REF]. The sample was incubated for 90 minutes at 37 o C. 5 µl of the reactions were spotted on DE81 paper for quantification of incorporated labelled nucleotides [START_REF] Maniatis | Molecular clonig: a laboratory manual[END_REF]. The rest of the reactions were stopped by adding EDTA, SDS and proteinase K to final concentrations of 20 mM, 0.65% and 1.7 µg/µl, respectively and a further 30 minutes of incubation. The sample was extracted once with phenol/chloroform and DNA was passed over a G-50 spin column (Boehringer / Mannheim) into TE buffer (10 mM Tris-HCl pH 8; 1 mM EDTA) to remove unincorporated nucleotides. DNA was ethanol precipitated in the presence of 10 µg carrier tRNA.

For the mono-polymerase system DNA was dissolved in 20 µl alkaline loading buffer (50 mM NaOH; 1mM EDTA; 5 % Ficoll 400; 0.025% bromcresol green). The samples were separated in 1.5% agarose gels in circulating alkaline running buffer (50 mM NaOH; 1mM EDTA) for 10 hr at 150 mA in the cold. The gel was fixed in 10 % trichlor acetic acid, dried and exposed to X-ray films. For SV40 DNA replication the DNA was dissolved in 20 µl TE buffer and 5 µl were double restricted with EcoR 1 to linearize the plasmid DNA and Dpn I to remove un-replicated DNA. Native loading buffer (20 mM Hepes-KOH pH 8; 1 mM EDTA, 2% sucrose, 0.01% bromophenol blue) was added and products were separated in a 0.8% agarose gel in TBE (89 mM Tris-borate, 89 mM boric acid). The gel was dried and exposed to X-ray films.

To analyse the effects of antibodies and peptides at different steps, ATP, other rNTPs and dNTPs were omitted in the initial sample to stall the mono-polymerase reaction before the unwinding, primer synthesis and primer elongation steps. Peptides or antibodies added to these reactions were pre-incubated with the proteins for 30 minutes prior to the release of the block by supplementing the missing nucleotides.

Enzmatic assays using natural single stranded DNA templates

Reactions were set up as for the mono-polymerase system with two changes: first Topo was omitted and second the SV40 origin containing DNA was replaced by 0.083 pmol ssM13mp18 DNA (2.4 µg/pmol) or ssM13mp18 DNA primed with the universal primer oligonucleotide (5'dGTAAAACGACGGCCAGT-3', Pharmacia) to serve as templates for primer synthesis and for primer extension, respectively [27].

For primer synthesis 0.2 mM of each GTP and UTP as well as 0.05 mM CTP along with 10 µCi α 32 P-CTP were added. Extension reactions were supplemented instead with 0.2 mM each dATP, dGTP and dTTP, 0.05 mM dCTP and 10 µCi α 32 P-dCTP.

Incubation proceeded for 90 minutes at 37 o C. Quantification and analysis of primer extension products were done as described for the mono-polymerase system. Products of the primer synthesis assays were ethanol precipitated in the presence of 0.8 M LiCl; 10 mM MgCl 2 and 10 µg carrier tRNA. After dissolving the sample in 20 µl denaturing loading buffer (35% formamide; 8 mM EDTA; 0.1% bromphenolblue; 0.1% xylene cyanol FF) for 30 min. at 65 o C one half of the samples were analysed in 20% denaturing urea polyacrylamide gels in TBE (0.089 M Tris-borate, 0.089 M boric acid, 0.01 M EDTA). Autoradiography was done with the wet gel.

RESULTS

Expression and characterization of peptides

We have expressed regions of Tag, Pol and RPA known to be involved in protein-protein interactions as soluble MBP-fusion peptides (see supplementary Figure 1). These included sequences spanning amino acids 164-249 for Tag (peptide T164-249) [START_REF] Weisshart | The replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication[END_REF], amino acids 195-313 for the p180 subunit of Pol (peptide P195-313) [START_REF] Dornreiter | Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen[END_REF] and amino acids 1-183 (peptide R1-173) and 174-250 (peptide R174-250) for the p70 subunit of RPA [START_REF] Braun | Role of proteinprotein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase α by multiple mechanisms[END_REF]. Peptides T164-249, P195-313 and R1-173 interacted specifically with the p32 and p70 subunits of RPA, Tag and the p180 subunit of Pol, respectively. R174-250 bound to full length Tag as well as the p48, p58 and p180 subunits of Pol (see supplementary Figure 2).

Since peptides associated specifically with certain replication factors, we next tested their ability to interfere with the interactions of the native proteins in co-precipitation assays.

Interference of peptides with protein-protein interactions

In the presence of peptides, specific associations between Tag, Pol and RPA were reduced in a dose-dependent manner (Figure 1). R174-250 diminished the amounts of RPA that were retained by either Tag (panel A) or Pol (panel B), whereas R1-173 slightly blocked the latter interaction. T164-249 specifically decreased the binding of Tag to RPA (panel C), but left the interaction with Pol uninfluenced (panel D). P195-313 inhibited only the binding of Pol to Tag (panel E), but not to RPA (panel F). MBP on its own induced no reduction in these mutual interactions. The amounts of peptides needed to see a pronounced competition effect were in the range of a 100 fold molar excess and higher over the intact factors.

We next investigated the effects the interference of protein-protein interactions exerted on DNA synthesis in the mono-polymerase system.

Interference of peptides with the mono-polymerase system

Peptides added at the beginning of mono-polymerase reactions inhibited incorporation levels compared to the control reaction in dependence of their concentration (Figure 2A). To see pronounced inhibition a 50, a 100 and a 500 fold excess over Tag were necessary for P195-313 (lanes 13-15), both T164-249 (lanes 6-8) and R174-250 (lanes 3-5), and R1-173 (lanes [START_REF] Stadlbauer | DNA replication in vitro by recombinant DNApolymerase-α-primase[END_REF][START_REF] Soe | A human topoisomerase I cleavage complex is recognized by an additional human topisomerase I molecule in vitro[END_REF][START_REF] Henricksen | Recombinant replication protein A: expression, complex formation, and functional characterization[END_REF], respectively. MBP had no effect on the reaction at either of the concentrations used (lanes 9-11). In addition, we tested other MBP-fusion peptides negative for inhibition. These included fusions with the smaller RPA subunits (MBP-p14 and MBP-p32), a sub-fragment of the largest subunit spanning amino acids 330-440 (MBP-p70.330-440) and cyclin A (MBPcyclin A) (data not shown). None of the peptides even at the highest concentrations used were able to diminish nucleotide incorporation to levels comparable to the negative control (lane 1). Back titration of up to 6 pmol Tag into assays containing peptide R174-250 relieved inhibition, restoring incorporation to levels obtained in the absence of the peptide (Figure 2B, We next determined the time points during the mono-polymerase reaction where proteinprotein interactions became important.

Influence of peptides on different steps of the mono-polymerase system

Peptides were pre-incubated with stalled reactions prior to the unwinding, primer-synthesis and primer extension steps by omitting ATP, other rNTPs and dNTPs from the reactions. All peptides were most potent if added prior to the unwinding step, when the template was a closed circular super-coiled substrate (Figure 2C, lanes U). When the peptides were supplied after the unwinding step and before the onset of primer synthesis with a partially unwound template, intermediate inhibition levels were observed (lanes I). When peptides were provided after the primer-synthesis step and before the onset of the primer extension reaction inhibition was almost completely lost and incorporation levels were comparable to the positive control (lanes E). MBP showed no influence on the elongation rates regardless of when it was added to the reaction (lanes 9-11).

In an extension to these results, we have also used other protocols to block the replication reactions at different time points (see supplementary methods and supplementary Figure 3). These data confirmed that the most pronounced inhibitory effect was seen as long as the peptides were administered prior to the primer synthesis step. Peptides added just prior to or after the primer extension reactions lost their inhibitory influence. The assembly of Tag onto the origin DNA and its bidirectional unwinding activities were only limited affected by the peptides.

The loss in interference at later replication steps could have been caused by two reasons.

First, the interactions between Tag, Pol and RPA were not essential for the primer extension step and following reactions, or second, peptides could not bind to these factors any more once primer synthesis was started. To discriminate between these possibilities we used monoclonal antibodies directed against the p70 subunit of RPA (70A, B and C) known to interfere with RPAs' ability to interact with the other replicative factors (for a characterization of the antibodies see supplementary Figure 4) [START_REF] Kenny | The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication[END_REF]. In a first step we tested the abilities of the antibodies to disrupt pre-formed complexes.

Interference of p70 specific monoclonal antibodies with protein-protein interactions

When 70A, B and C antibodies were pre-incubated with immobilized H 6 -RPA, its ability to interact with Tag and Pol was almost completely abolished (Figures 3A andB, lanes 2-4). In contrast, RAC antibodies directed against the C-terminal part of p70 still allowed RPA to bind as efficiently as in the control reaction without antibodies (compare lanes 6-7 with lane 1). A polyclonal serum proved somewhat inhibitory (lane 5). When 70A, B and C antibodies were added to the reaction after the replication factors were allowed to bind to each other, they efficiently disrupted the pre-formed interactions and little of the soluble protein was retained by H 6 -RPA (shown for 70B and C in lanes 8-9).

We next tested the influence of antibodies on the different steps of the mono-polymerase system.

Interference of p70 specific monoclonal antibodies with different steps of the monopolymerase reaction

Antibodies 70A, B and C inhibited SV 40 in vitro DNA replication (Figure 4A, lanes 7-12). 70C was most effective, abolishing the incorporation of nucleotides to levels comparable to the negative control without RPA (compare lane 4 to lane 1). Compared to positive controls (no antibody, lane 1; buffer, lane 2; and non-inhibitory Tag specific antibody PAb 101, lane 7), 70A and 70B also significantly decreased incorporation levels.

When antibodies were supplied after the unwinding step and before starting primer synthesis with addition of rNTPs (Figure 4B) or after the priming step and before primer extension with the addition of dNTPs (Figure 4C) in mono-polymerase assays, all three significantly reduced incorporation levels compared to the controls (compare lanes 4-6 to lanes 2-3, 7). As was the case in the in vitro replication assay, 70C was the most powerful inhibitory antibody. Please note that the antibody 70C required a 30 minute pre-incubation period before the next reaction step was activated in order to exert strong inhibition, whereas both 70A and 70B inhibited without the requirement of a pronounced pre-incubation phase (data not shown).

To assess the significance of protein-protein interactions to a greater extent, antibodies were tested using un-primed and primed ssM13mp18 DNA as templates. These substrates do not require any unwinding steps, making it possible to study the influence of antibodies directly on the stimulatory or inhibitory effects between the replication factors.

Influence of p70 specific monoclonal antibodies on RNA and DNA synthesis using natural single-stranded templates

Using un-primed ssM13mp18 DNA as a template, no primer synthesis took place in the absence of Pol (Fig 5A ,lane 1). In its presence, primers 2 -25 nucleotides in length were synthesized (lane 2). The presence of RPA inhibited primer synthesis (lane 3), whereas Tag led to an increase in the synthesis rate (lane 4). In addition, the inhibition induced by RPA was overcome in the presence of Tag (lane 5). In the absence of Tag, antibodies 70A and B left incorporation levels nearly uninfluenced, at most a slight decrease could be noticed (compare lanes 7 and 8 to lane 3). In contrast, 70C lead to a slight stimulation of the primer synthesis reaction (compare lane 9 to lane 3). In the presence of Tag, all three antibodies reduced incorporation rates (compare lanes 9-11 to lane 5). 70C was hereby least efficient.

Using a primed ssM13mp18 DNA template, Pol synthesized products 80-250 nucleotides long (Figure 5B, lane 2). In its absence no synthesis was observed (lane 1). In this setting RPA and Tag stimulated polymerase activity, with RPA being more potent (lanes 3 and 4).

Intermediate stimulation was seen in the presence of both proteins, indicating that Tag decreased the stimulatory effect of RPA to some extent (lane 5). In the absence or presence of Tag, all antibodies inhibited DNA synthesis, with 70C being significantly least efficient (compare lanes 6-8 and 9-11 to lane 3).

The influence of some of the interactions on enzymatic activities was also substantiated using the peptides (see supplementary Figure 5). Interference between the Tag and Pol interaction by peptide P195-313 lead to a decrease on the stimulatory effect of Tag on both the primer synthesis and primer extension reactions catalyzed by Pol. Interfering with the Pol-RPA interaction lead to a decrease in the efficiency of Pol to extend primers, but had no effect on the inhibition of primer synthesis exerted by RPA. Lastly, disturbing the interaction between Tag and RPA led to a decrease in Tag's ability to reverse the inhibitory and stimulatory effects of RPA on primer synthesis and primer extension, respectively.

DISCUSSION

Replication of the SV40 mini-chromosome in cell free extracts starts when the virally encoded T-antigen (Tag) binds to the origin sequences as a double hexameric complex [START_REF] Simmons | SV40 large T antigen functions in DNA replication and transformation[END_REF]. In subsequent steps Tag marshals the association of three cellular factors, replication protein A (RPA) [START_REF] Dornreiter | Interaction of DNA polymerase α-primase with cellular replication protein A and SV40 T antigen[END_REF][START_REF] Melendy | An interaction between replication protein A and SV40 T antigen appears essential for primosome assembly during SV40 DNA replication[END_REF], DNA polymerase α-primase (Pol) [START_REF] Dornreiter | Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen[END_REF][START_REF] Murakami | Functional interactions between SV40 T antigen and other replication proteins at the replication fork[END_REF] and topoisomerase I (Topo) [START_REF] Roy | The cap region of topoisomerase I binds to sites near both ends of simian virus 40 T antigen[END_REF][START_REF] Trowbridge | Human topoisomerase I promotes initiation of simian virus 40 DNA replication in vitro[END_REF][START_REF] Seinsoth | Bidirectional DNA unwinding by a ternary complex of T antigen, nucleolin and topoisomerase I[END_REF] to generate the initiation complex. After the initiation phase provides RNA-DNA primers for the leading and lagging strand DNA synthesis, full replication is accomplished by additional cellular factors [START_REF] Waga | Reconstitution of complete SV40 DNA replication with purified replication factors[END_REF]. Here we studied the significance of the physical interactions between Tag, Pol and RPA during different steps in the initiation reactions by using inhibitory peptides and antibodies.

The employed peptides and antibodies were able to interfere or disrupt specific interactions between Tag, RPA and Pol (Figures 1 and3), but had otherwise no influence on replication activities of these factors (data not shown and supplementary Figures 1 and3). Only antibody 70C, which binds close to DBD-A of the p70 subunit of RPA showed a destabilization of the RPA-DNA complex and as a consequence interfered with Tag-catalyzed bidirectional unwinding, which needs an ssDNA binding activity [START_REF] Braun | Role of proteinprotein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase α by multiple mechanisms[END_REF][START_REF] Kenny | The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication[END_REF][START_REF] Brill | Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication[END_REF]. Antibodies 70A and B, which bind at the N-terminal part of RPA, were very potent in blocking the formation of proteinprotein contacts and disrupted preformed complexes, probably due to steric hindrance. This possibility is consistent with the observation that both antibodies inhibit Pol stimulation by RPA [START_REF] Kenny | The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication[END_REF]. Surprisingly, antibody 70C was reported not to interfere with Pol stimulation [START_REF] Kenny | The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication[END_REF], albeit it interfered strongly with Pol binding in our hands. However, this antibody needed a prolonged pre-incubation step with RPA in order to exert its inhibitory effects in enzymatic assays, and therefore its negative influence might easily have escaped observation.

In contrast to antibodies, peptides were markedly selective in blocking association reactions (Figure 1). P195-313 and T164-249 interfered solely with Tag's and RPA's ability to interact with those partner molecules for which they mimicked the binding interface, which are Pol and Tag [START_REF] Weisshart | The replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication[END_REF][START_REF] Dornreiter | Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen[END_REF]. R1-173, on the other hand, exclusively interfered with the docking of Pol on RPA, consistent with the idea that the corresponding sequences on the p70 subunit are important for Pol stimulation [START_REF] Kim | Role of the 70-kDa subunit of human replication protein A (I). Single-stranded DNA binding activity, but not polymerase stimulatory activity, is required for DNA replication[END_REF][START_REF] Braun | Role of proteinprotein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase α by multiple mechanisms[END_REF]. R174-250, representing a sequence within DBD-A, blocked access of both Tag and Pol, emphasizing the role of this domain in the control of Pol's processivity, which is positively and negatively regulated by RPA and Tag, respectively [START_REF] Braun | Role of proteinprotein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase α by multiple mechanisms[END_REF]. The presence of the peptides at the onset of the reaction inhibited the incorporation levels in the mono-polymerase system in a concentration dependent manner (Figure 2). The huge peptide excess over replication factors needed to exert significant inhibition indicate that the peptides in all probability do not contain the full set of amino acids involved in forming contact sites between the full length proteins. In addition, some peptides like R1-174 might only interfere with the initial docking of the interacting molecules, but might not block their major binding sites (see supplementary Figure 2). The inhibitory effect of the peptides could be overcome by adding back the respective replication factor, corroborating the interpretation that interferences were due to disturbances in protein-protein interactions.

Kinetics of inhibition showed that the peptides were most effective if present before the unwinding step, which itself was only slightly affected. Inhibition capacity was reduced if peptides were added after unwinding and before primer synthesis and ceased completely when they were supplemented after the primer synthesis and before the primer extension reaction. This suggests that the peptides can form unproductive complexes with replication factors only during the early steps in the initiation reaction. Our data indicate that interactions were transient during the assembly step of Tag onto the origin DNA (supplementary Figure 5). They became meta-stable during the unwinding reaction, when the pre-initiation complex is assembled. Once Pol was engaged in primer synthesis, a stable initiation-complex or primosome was formed that acted in a processive manner. The fact that complex disruption by antibodies could still interfere with primer synthesis and extension at these late stages shows the importance of the physical interactions between Tag, RPA and Pol throughout the initiation process.

The significance of mutual interactions on enzymatic activities was additionally demonstrated in inhibition studies using natural single-stranded DNA templates (Figure 5 and supplementary Figure 5). Primer synthesis on circular single-stranded DNA templates by Pol was greatly reduced in the presence of RPA. Interference with the RPA-Pol interaction did not relief the block suggesting that the inhibition is indirectly exerted by RPA by blocking Pol's access to the template [START_REF] Melendy | An interaction between replication protein A and SV40 T antigen appears essential for primosome assembly during SV40 DNA replication[END_REF][START_REF] Collins | The effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase α-primase[END_REF][START_REF] Murakami | Studies on the initiation of simian virus 40 replication in vitro: RNA primer synthesis and its elongation[END_REF]. Such an occlusion effect was corroborated by a slight stimulatory effect of antibody 70C, which destabilizes the DNA-RPA complex.

Inhibition was overcome in the presence of Tag, but the latter's stimulatory effect was abrogated to a great extent or diminished by all antibodies and by those peptides that inhibited Tag's ability to bind to RPA. This suggests that Tag in cooperation with Pol can alter the DNA binding properties of RPA through direct interactions [START_REF] Jiang | Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex[END_REF]. In respect to the antibodies 70C was less potent in its inhibitory power than 70A and B, probably because the destabilizing influence on DNA binding reversed the negative effect exerted by the disruption of protein-protein interactions to a certain level. A primed template, on the contrary, was a better substrate for Pol when coated with RPA and a strong stimulation in the polymerisation activity could be observed compared to a naked primed circular ssDNA substrate [START_REF] Kim | Role of the 70-kDa subunit of human replication protein A (I). Single-stranded DNA binding activity, but not polymerase stimulatory activity, is required for DNA replication[END_REF][START_REF] Braun | Role of proteinprotein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase α by multiple mechanisms[END_REF][START_REF] Kolpashchikov | Interaction of the p70 subunit of RPA with a DNA template directs p32 to the 3'-end of nascent DNA[END_REF][START_REF] Kolpashchikov | Polarity of human replication protein A binding to DNA[END_REF]. In this setting RPA seemed to block unproductive binding sites on the template and to actively stabilize Pol on the primer-template junction, since all antibodies and peptides inhibiting the Pol-RPA interaction abolished the stimulatory effect to a great or some extent, respectively. In addition, the clearance of RPA that is required during polymerization might be due to Pol-RPA interactions. 70C with its destabilizing effect on RPA-DNA complexes would facilitate the release of RPA, further accounting for its lower inhibition capacity compared to the other two inhibitory antibodies 70A and 70B. Tag was stimulatory to both the priming and polymerisation activity of Pol using the naked primed template, underlining the importance of its interaction with the p180 subunit of Pol. Indeed, peptides inhibitory for the Pol-Tag interaction reduced this positive effect in both settings [START_REF] Dornreiter | SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha[END_REF][START_REF] Dornreiter | Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen[END_REF]. On the other hand, the presence of Tag reduced incorporation levels on the RPA covered substrate. This might have been due to a stabilizing effect on the DNA binding of RPA, since the antibody 70C could reverse this inhibition to a great extent and interference with the Tag-RPA interaction by peptide T164-249 also resulted in a stimulation of the extension reaction.

Experiments to explain the precise assembly of the initiation complex that culminates in the synthesis of RNA primers and their extension with DNA are ongoing [START_REF] Arunkumar | Insights into hRPA32 C-terminal domain-mediated assembly of the simian virus 40 replisome[END_REF][START_REF] Simmons | Assembly of the replication initiation complex on SV40 origin DNA[END_REF]. Inhibition studies like the one presented in this report should be helpful to understand the dynamics of the replication process in terms of the timed formation of protein-protein as well as protein-DNA interactions in future work.

FIGURE LEGENDS

  and T96.249C for peptide T164-249 (Tag); and P180.195N and P180.313C for peptide P195-313 (Pol). The numbers preceding "N" and "C" indicate the first N-and last C-terminal expressed amino acids of the resulting amplified sequences. The primers had the following sequences with restriction sites used for cloning underlined: R70.1N: 5'-TTGGATCCCATATGGTCGGCCAGCTGAGCGA-3'; R70.174N: 5'-TTGGATCCTCACACACTTCTGGGGGAA-3'; R70.173C: 5'-TTGAATTCTCACAGGCTGGGACCTGCAG-3'; R70.250C: 5'-TTGAATTCTCACACTTCAATAAGAGGAAAGAACT-3'; T96.164N: 5'-TTGGATCCCATATGAAGGAAAAAGCTGCACT-3'; T96.249C: 5'-TTGGATCCTCATGGCAAACTTTCCTCAA-3' P180.195N: 5'-TTGAATCCCATATGTCTGTGCACACCGCCAC-3' P180.313C: 5'-TTGGATCCTCAACCTTCTTGATCAATGT-3' The EcoRI/BamHI fragments were cloned into pBluescript KS II+ (Stratagene) to yield plasmids pBS(N)/RPA70(1-173), pBS(N)/RPA70(174-250); pBS(N)/TAG(164-249), and pBS(N)/POL(195-313) respectively and were confirmed by sequencing. The BamHI-SalI fragments were then sub-cloned into pMAL-c2 (New England Biolabs) to yield plasmids pMAL/RPA70.1-173, pMAL/RPA70.174-259; pMAL/TAG96.164-249, and pMAL/POL180.195-313.

  compare lanes 3-6 to lane 2). Increasing the amounts of Tag beyond 6 pmol led to a reduction of incorporation rates (lanes 7-8), suggesting quenching of other factors by this excess of Tag. Likewise the inhibition of P195-313 and T164-249 could be reduced by addition of extra Tag (lanes 11-12) and RPA (lanes 13-14), respectively. Addition of the same amounts of these two factors to reactions without peptide showed only a minor stimulatory effect (data not shown). The addition of extra Pol increased incorporation levels in the presence of R1-173 (lanes 9-10), but stimulated the reaction to the same relative extent in the peptide's absence (data not shown).

Figure 1 :

 1 Figure 1: Inhibition of protein-protein interactions by peptides. Pair wise co-precipitations were performed between Tag and RPA (A, C), Pol and RPA (B, F), and Pol and Tag (D, E), each at 25 pmol. The immobilized bait proteins are indicated by the antibody symbol on the left. Co-precipitated target proteins Tag (p96), Pol (p180 subunit)and RPA (p70 subunit) were revealed by western blotting using the enhanced chemiluminescence detection system. Peptides were present at a 25 and 100 fold molar excess to the target protein or were omitted (0). The numbers below each lane represent the average pmol of co-precipitated protein as determined by densitometric scanning from 5 independent experiments. The position of the heavy chain of immunoglobulin (IgH) is indicated when it was detected due to cross-reactivity of the secondary antibodies. I, 1/10 th of the input material of soluble target proteins; NA, no antibody added.

Fig. 2 :

 2 Fig. 2: Influence of peptides on the mono-polymerase system. Panel A. Titration of peptides. Increasing amounts of indicated peptides displayed as fold molar excess over 6.25 pmol Tag were titrated into the reaction at the onset of monopolymerase reactions. Panel B. Back-titration of replication factors. Replication factors were added back in the indicated amounts at the onsets of the reactions. Molar excess of peptides over Tag was 100 fold (for R1-173 500 fold). Panel C. Kinetics of inhibition. Indicated peptides were added to the reaction at a 100 fold (500 fold for R1-173) molar excess over Tag before unwinding (U), primer synthesis (I) and primer elongation (E).The different template configurations present at the time points the peptides were administered were under-wound (U), primed (I) and primer extended (E) plasmid DNAs, which are symbolized for each reactions. Lanes N and P represent negative and positive controls in the absence and presence of Tag. The numbers below lanes denote the incorporated pmol of nucleotides.

Fig. 3 :

 3 Fig. 3: Influence of p70 specific monoclonal antibodies on protein-protein interactions. H 6 -RPA was linked to beads via anti-his antibodies. The resin was incubated with the indicated antibodies before (pre) or after (post) addition of the other replication factors. Anti-RPA refers to a polyclonal serum. Co-precipitated proteins were detected by western blotting using the enhanced chemiluminescence detection system. The positions of Tag (p96) and the p180 subunit of Pol are indicated. Lane 1 contained 1/10 th of the input material of the soluble target protein. Numbers below each lane indicate the amounts of co-precipitated proteins in pmol as determined by densitometry.

Fig. 4 :

 4 Fig. 4: Influence of p70 specific antibodies on replication related activities of RPA. Antibodies were tested in SV40 DNA replication (panel A), primer synthesis (panel B), and primer extension (panel C) assays. In panel A each DNA was restricted with EcoRI alone (E, odd numbered lanes) or double restricted by EcoRI-DpnI (D, even numbered lanes). The numbers below lanes indicate the pmol of incorporated dNMPs (A and C) or NMPs (B). Lane 1 contained no protein. The positions of the products of each reaction, linear dsDNA (form II), RNA-primers (RP) and RNA-DNA primers (RDP), are indicated. Markers contained oligo(dT) 8 -12 or an 81 base pair fragment. Antibodies were present at the onset of the reaction (A) or before the unwinding (B) or primer-synthesis (C) steps. The template configurations present at the time point antibodies were administered were super-coiled, under-wound and primed plasmid DNAs and are represented by symbols for each reaction.

Fig. 5 :

 5 Fig. 5: Influence of antibodies on primer synthesis and extension using natural singlestranded DNA templates. Primer synthesis (panel A) and primer extension (panel B) reactions were carried out using an un-primed or primed single stranded circular DNA, respectively, as the templates, which are indicated by symbols for each reaction. The presence of any of the replication proteins Tag, Pol and RPA are indicated by "+", their absence by "-". Antibodies were added as specified (lanes 6-11). The numbers below each lane indicate the incorporated pmol of rNMPs (A) and dNMPs (B). RNA (RP) and DNA (DP) products are indicated.

Figures

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Biochemical

  Journal Immediate Publication. Published on 1 Aug 2007 as manuscript BJ20070794 © 2007 The Authors Journal compilation © 2007 Biochemical Society

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Biochemical Journal Immediate Publication. Published on 1 Aug 2007 as manuscript BJ20070794 © 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENT

This work was supported by NIH (GM52948 to EF, C00004284-1 to HPN), Vanderbilt University (to EF), Deutsche Forschungsgemeinschaft (SFB604 to FG), Health Research Board Ireland (RP/2004/154 to HPN) and Science Foundation Ireland (05/RFP/BIC0022 to HPN). The Leibniz Institute for Age research is financially supported by the federal government and the Land Thueringen.