Supplementary Materials Supplementary Methods, Supplementary Figures 1S-5S and results bj4070313add. RPA

Supplementary Materials Supplementary Methods, Supplementary Figures 1S-5S and results bj4070313add. RPA and Pol were replaced by 190?g of S100 extracts obtained from HEK-293 cells (human embryonic kidney cells) [25]. The sample was incubated for 90?min at 37?C. A 5?l volume of each reaction combination was spotted on DE81 paper for quantification of incorporated labelled nucleotides [26]. The reactions were stopped by adding EDTA, SDS and proteinase K to final concentrations of 20?mM, 0.65% and 1.7?g/l respectively and a further 30?min 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, and 1?mM EDTA) to remove unincorporated nucleotides. DNA was ethanol-precipitated in the presence of 10?g of carrier tRNA. For the monopolymerase system, DNA was dissolved in 20?l of alkaline loading buffer [50?mM NaOH, 1?mM EDTA, 5% (w/v) Ficoll 400 and 0.025% Bromcresol Green]. The samples were separated in 1.5% agarose gels in circulating alkaline running buffer (50?mM NaOH and 1mM EDTA) for 10?h at 150 mA in the cold. The gel was fixed in 10% (v/v) trichloroacetic acid, dried and exposed to X-ray films. For SV40 DNA replication, the DNA was dissolved in 20?l of TE buffer, and 5?l was double-restricted with EcoRI to linearize the plasmid DNA and DpnI to remove un-replicated DNA. Native loading buffer [20?mM Hepes/KOH, pH?8, 1?mM EDTA, 2% (w/v) sucrose and 0.01% Bromophenol Blue] was added, and products were separated in a 0.8% agarose gel in TBE (Tris/borate/EDTA; 89?mM Tris/borate, 89?mM boric acid and 10?mM EDTA). The gel Azacitidine supplier was dried and exposed to X-ray films. To analyse the effects of antibodies and peptides at different actions, ATP, other rNTPs and dNTPs were omitted in the initial sample to stall the monopolymerase reaction before the unwinding, primer synthesis and primer extension actions. Peptides or antibodies added to these reactions were pre-incubated with the Mouse monoclonal to Myoglobin proteins for 30?min prior to the release of the block by supplementing the missing nucleotides. Enzymatic assays using natural ssDNA templates Reactions were set up as for the monopolymerase system with two changes: first, Topo was omitted and, secondly, the SV40 origin containing DNA was replaced by 0.083?pmol of ssM13mp18 DNA (2.4?g/pmol) or ssM13mp18 DNA primed with the universal primer oligonucleotide (5-dGTAAAACGACGGCCAGT-3; GE Healthcare) to serve as templates for primer synthesis and for primer extension respectively [27]. For primer synthesis, 0.2?mM each of GTP and UTP and also 0.05?mM CTP along with 10?Ci of [-32P]CTP were added. Extension reactions were supplemented instead with 0.2?mM each of dATP, dGTP and dTTP, 0.05?mM dCTP and 10?Ci of [-32P]dCTP. Incubation proceeded for 90?min at 37?C. Quantification and analysis of primer extension products were performed as explained for the monopolymerase system. Products of the primer synthesis assays were ethanol-precipitated in the presence of 0.8?M LiCl, 10?mM MgCl2 and 10?g of carrier tRNA. After dissolving the sample in 20?l of denaturing loading buffer [35% (v/v) formamide, 8?mM EDTA, 0.1% Bromophenol Blue and 0.1% Xylene Cyanol FF] for 30?min at 65?C, one-half of the samples were analysed in 20% denaturing urea/polyacrylamide gels in TBE. Autoradiography was performed with the wet gel. RESULTS Expression and characterization of peptides We have Azacitidine supplier expressed regions of Tag, Pol and RPA that are known to be involved in proteinCprotein interactions as soluble MBP-fusion peptides (see Supplementary Physique 1 at These included sequences spanning proteins Azacitidine supplier 164C249 for Tag (peptide T164-249) [9], proteins 195C313 for the p180 subunit of Pol (peptide P195-313) [28] and proteins 1C183 Azacitidine supplier (peptide R1-173) and 174C250 (peptide R174-250) for the p70 subunit of RPA [8]. Peptides T164-249, P195-313 and R1-173 interacted particularly with the p32 and p70 subunits of RPA, Tag and the p180 subunit of Pol respectively. Peptide R174-250 bound to full-duration Tag and also the p48, p58 and p180 subunits of Pol (see Supplementary Body 2 at Since peptides linked specifically with specific replication elements, we following tested their capability to hinder the.