Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile

DNA replication is an essential and conserved process in all domains of life and may serve as a target for the development of new antimicrobials. However, such developments are hindered by subtle mechanistic differences and limited understanding of DNA replication in pathogenic microorganisms. Clostridium difficile is the main cause of healthcare-associated diarrhoea and its DNA replication machinery is virtually uncharacterized. We identify and characterize the mechanistic details of the putative replicative helicase (CD3657), helicase-loader ATPase (CD3654) and primase (CD1454) of C. difficile, and reconstitute helicase and primase activities in vitro. We demonstrate a direct and ATP-dependent interaction between the helicase loader and the helicase. Furthermore, we find that helicase activity is dependent on the presence of primase in vitro. The inherent trinucleotide specificity of primase is determined by a single lysine residue and is similar to the primase of the extreme thermophile Aquifex aeolicus. However, the presence of helicase allows more efficient de novo synthesis of RNA primers from non-preferred trinucleotides. Thus, loader–helicase–primase interactions, which crucially mediate helicase loading and activation during DNA replication in all organisms, differ critically in C. difficile from that of the well-studied Gram-positive Bacillus subtilis model.

antifoam 204 (Sigma-Aldrich), was inoculated with a pre-culture (10 mL). The cell culture was incubated at 37°C with mechanical shaking at 180 rpm, until an optical density (600 nm) of 0.70-0.85 was reached (after approximately 3 h). Protein expression was induced via the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG; 1 mM) and the culture was incubated at 30 °C for 3 h. The cells were harvested by centrifugation (3000 g, 15 min, 4 °C) and the resulting cell paste was stored at -80°C.
CD3657 cell paste, prepared from 1.2 L cell culture, was re-suspended in 30 mL TED50 buffer (Tris pH 7.5 50 mM, EDTA 1 mM, DTT 1 mM, NaCl 50 mM) with PMSF (1 mM). The bacterial cells were lysed by sonication and crude lysate was clarified by centrifugation (35,000 g, 30 min, 4°C). The resulting supernatant was separated from the cell debris using a 0.22 μm pore filter before a 50% w/v ammonium sulphate precipitation, followed by clarification by centrifugation (35,000 g, 30 min, 4°C). The ammonium sulphate precipitated pellet was suspended in TED50 buffer and loaded onto a 5 mL Q sepharose column, equilibrated in TED50 buffer. The protein was eluted using a gradient of 20 to 100% TED1000 buffer (Tris pH 7. The protein was quantified by UV spectrophotometry and stored at -80°C. Protein purity (>95%) was estimated by SDS-PAGE electrophoresis and concentration was determined spectrophotometrically using extinction coefficients calculated using the ExPASy ProtParam tool (http://web.expasy.org/protparam).
Walker A mutant proteins (K214R and T215A) were purified in a manner identical to the wild-type proteins after overexpression of the protein from the plasmids pEVE90 and pEVE92, respectively.
Purification of the putative loader protein CD3654 C. difficile CD3654 was expressed from the pEVE-24 plasmid in E. coli BL21 (DE3). The growth medium, consisting of 2xYT broth (1 L), kanamycin (30 μg/mL) and antifoam 204 (Sigma-Aldrich), was inoculated with a pre-culture (10 mL). The cell culture was incubated at 37 °C with mechanical shaking, until an optical density (600 nm) of 0.62-0.65 was reached (after approximately 3 h). Protein expression was induced via the addition of IPTG (1 mM) and the culture was incubated at 30 °C for 3 h. The cells were harvested by centrifugation (3000 g, 15 min, 4 °C and the resulting cell paste was stored at -80 °C. The bacterial cell paste, prepared from 1 L cell culture, was suspended in 25 mL TED50 buffer with PMSF (1 mM) and protease inhibitor cocktail (100 μL). The cells were lysed by sonication, clarified by centrifugation (40,000 g, 30 min, 4 °C) and the resulting supernatant separated from the cell debris using a 0.22 μm pore filter. Ammonium sulphate (7.32 g) was added slowly to the supernatant (25 mL) with stirring at 4 °C, to achieve 49% saturation. The protein pellet was collected by centrifugation (40,000 g, 30 min, 4 °C) and washed with TED20 buffer (Tris pH 7.5 50 mM, EDTA 1 mM, DTT 1 mM, NaCl 20 mM) (2 x 4 mL). The precipitate was suspended in TED20 buffer (15 mL) with gentle mechanical shaking (30 min, 4 °C). The suspension buffer was exchanged by dialysis against 1 L TED20 buffer for 2 h at 4 °C, giving a solution with conductivity of 9.5 mS. The protein solution (ca. 15 mL) was loaded onto combined 5 mL Q sepharose and 5 mL SP sepharose columns connected in series and equilibrated in TED20 buffer; the protein of interest eluted in the flow through. The collected protein was loaded onto a 5 mL heparin sepharose column equilibrated in TED20 buffer, and stepwise eluted to 15% TED1000 buffer. The collected protein was loaded onto a Hiload 26/60 Superdex 200 gel filtration column equilibrated in storage buffer (Tris pH 7.5 50 mM, EDTA 1 mM, DTT 1 mM, NaCl 50 mM, 10% v/v glycerol).
The protein was quantified by UV spectrophotometry and stored at -80 °C. Protein purity (>95%) was estimated by SDS-PAGE electrophoresis and concentration was determined spectrophotometrically using extinction coefficients calculated using the ExPASy ProtParam tool (http://web.expasy.org/protparam).
Walker A (K198R, T199A) and B (D258Q) mutant proteins were purified in a manner identical to the wild-type proteins after overexpression of the protein from the plasmids pEVE59, pEVE60 and pEVE203, respectively.

Purification of the primase CD1454
Overexpression of C. difficile CD1454 was carried out in E. coli BL21 (DE3) from the pEVE7 plasmid. The growth medium consisting of 2xYT broth (1.2 L), carbenicillin (50 μg/mL) and antifoam 204 (Sigma-Aldrich) was inoculated with a pre-culture (10 mL). The cell culture was Protein purity (>95%) was estimated by SDS-PAGE electrophoresis and concentration was determined spectrophotometrically, using extinction coefficients calculated using the ExPASy ProtParam tool (http://web.expasy.org/protparam).
The lysine mutant CD1454 (K70H) was purified in a manner identical to the wild-type proteins after overexpression of the protein from plasmid pEVE201.

Gel-filtration experiments
Self-interaction of the CD3657 and CD3654 proteins were studied in the presence and absence of ATP. In brief, purified CD3657 (or mutant) or CD3654 (or mutant) was incubated for 10 min at room temperature with MgCl 2 (2 mM) in their storage buffer and ATP (1 mM). The mixture Standards 151-1901, BioRad) were run to allow MW estimation. The column resolved the largest protein (670kDa) from this standard as a peak at an elution volume of ~9mL.
To assess interactions between CD3657 and CD3654, purified proteins were mixed in a 1:1 stoichiometry in the presence of MgCl 2 (2 mM) and ATP (1 mM), and incubated for 10 min at room temperature. The mixture (500 μL) was loaded onto a Hiload 10/300 GL Superdex 200 analytical grade size exclusion column equilibrated in buffer A at a flow rate of 0.5 mL/min. For the experiments without ATP, buffer B was used.
Samples from fractions were analysed by SDS-PAGE and Coomassie Blue staining to verify the identity of the proteins.

Bacterial two-hybrid assays
To determine (self-) interaction, pKEK1286-and pKEK1287-derived constructs were subsequently transformed in to the E. coli reporter strain KDZif1ΔZ [3,4]. In order to control for background due to (possible) differences in expression of the constructs, single expression plasmids (Zif or ω fusion) containing the gene of interest were transformed into the reporter strain. After overnight incubation at 30˚C, three colonies per assay were cultured overnight in LB broth (30°C) in the presence of 1mM IPTG, tetracycline (selects Zif fusion plasmid) and/or ampicillin (selects ω fusion plasmid). Bacterial cells were permeabilized with SDS and chloroform and assayed for β-galactosidase activity. In short, cells were diluted in Z buffer (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCl, 1 mM MgSO 4 , 50 mM β-mercaptoethanol, pH7 ) to 1 ml and permeabilized with 50 μl 0.1% w/v SDS and 100 μl chloroform. After 5 minutes of Van Eijk, Paschalis, Green et al 10 equilibration, 200 μl of o-nitrophenyl-β-D-galactopyranoside was added to each tube and incubated at room temperature until yellow colour developed. The reaction was stopped with 0.5 ml 1 M Na 2 CO 3 and measured at OD 420nm and OD 550nm to calculate the β-galactosidase activity in Miller Units. Experiments were performed in triplicate.

Helicase assays
Helicase activity was assayed by monitoring (and quantifying) the displacement of the radiolabelled (γ 32 P-ATP) oligonucleotide oVP-1 (partially) annealed to the single stranded circular DNA m13mp18 (ssM13; Affymetrix) essentially as previously described [5]. In short, the 105-mer oligonucleotide was radiolabelled at the 5' end using γ 32 P-ATP and T4 polynucleotide To investigate the effect of the putative helicase loader CD3654 or primase CD1454 on the activity of the helicase CD3657, the proteins were mixed in equimolar concentrations (1 µM) and incubated for 5 minutes at 37˚C before initiating the reaction with 2.5 mM ATP (final concentration). For three protein reactions, the buffer with CD3657 was preincubated for 5 mins before the addition of CD3654, then further preincubated for 5 mins 37˚C , followed by the addition of CD1454 and further preincubation, prior to initiation of the reaction with 2.5 mM ATP.
Stop buffer was added to terminate the reactions (1% v/w SDS, 40 mM EDTA, 8% v/v glycerol, 0.1% w/v bromophenol blue). Reaction samples (10 µl) were loaded on a 10% nondenaturing polyacrylamide gel, run in 1xTBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA) at 150V, 40mA/gel for 60 mins. The gel was dried, scanned and analyzed using a molecular imager and associated software (Biorad). Experiments were carried out in triplicate, and data analysis was performed using Prism 6 (GraphPad Software).

Size exclusion chromatography coupled with multiangle laser light scattering analysis
The oligomeric state of CD1454 protein was assessed using an 1260 Infinity HPLC system (Agilent), with a miniDAWN Treos 3-angle static light scattering detector (Wyatt Technologies) and ERC RefractoMax 521 UV 280nm and refractive index detector (ThermoScientific), connected downstream of a Superose 6 10/300 gel filtration column (GE Healthcare) equilibrated in 50 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM ATP, 2 mM MgCl 2 . The data was processed using the Astra™ package version 6.1 (Wyatt Technologies).

RNA primer synthesis assay and thermally denaturing HPLC analysis
RNA priming assays and denaturing HPLC analyses were conducted as was previously described for other mesophilic bacterial primases [6]. Initially, two 50-mer oligonucleotides that comprised all 64 trinucleotide sequences were used to assess enzymatic activity and template specificity, as previously described [7]. Confirmation of the preferred initiation motif was