Expression vector pBAD/HisA and E. coli TOP10 competent cells were obtained from Invitrogen (Groningen, NL). DpnI was obtained from New England Biolabs (Herts., UK). Y. pestis was manipulated using suitable biological safety precautions and containment facilities. Y. pestis genomic DNA was isolated from Y. pestis strain GB using a PureGene DNA isolation kit from Gentra Systems (Minneapolis, USA). Other DNA was isolated from cultures or agarose gels using commercially available kits following the manufacturers instructions. E. coli GM215 was obtained from the Yale E. coli genetic stock centre (CGSC# 6645). PCR primers were purchased from Sigma-Genosys (Haverhill, UK). For fluorescence measurements, oligonucleotides 1 and 2 were purchased from ATDBio (Southampton, UK), flat bottomed black 96 well polypropylene and half area flat bottomed 96 well polystyrene microplates were purchased from Greiner Bio-One Ltd (Stonehouse, UK) and measurements were taken using a Tecan Safire 2 microplate reader (Reading, UK). Assays for high throughput screening were prepared using a Beckman Coulter Biomek 3000 liquid handling system equipped with a 200 µl single channel and 20 µl 8 channel pipette head. Mineral oil (sterile filtered, mouse embryo tested, light oil) and S-adenosylmethionine chloride were obtained from Sigma Aldrich (Poole, UK). Bovine serum albumin (BSA) was purchased from Advanced Protein Products Ltd (Brierley Hill, UK).
Construction of pRJW4213/07 for the expression of Y. pestis Dam
DNA manipulations were carried out using standard protocols 
. The dam
gene was amplified by PCR from Y. pestis
GB genomic DNA using Pfu
Turbo polymerase and the following oligonucleotide primers: pfdam
AAGAAAAACCGCGCTTTTTTAAAATGG and prdam
TCAGCTATAGAGCGCCAAAAG. The dam
gene was amplified adding an NcoI site (italics) and DNA encoding a His6
tag (bold) to the 5′ end of the amplified product, and a HindIII site (italics) at the 3′ end. The PCR amplification product was purified and the NcoI-HindIII fragment containing the modified dam
gene inserted by ligation between unique NcoI and HindIII sites of pBAD/HisA to yield plasmid pRJW4213/07.
Expression and purification of Y. pestis Dam
All purification steps were carried out at 4°C and samples centrifuged in a Beckman JA-14 rotor unless otherwise stated. All cell culture media contained 100 µg/ml ampicillin. Cell pastes and purified proteins were stored at −80°C. Protein purity was judged by SDS-PAGE with Coomassie staining.
2YT medium (100 ml) was inoculated from stored strains (pRJW4213/07 in E. coli strain GM215) and grown overnight in a shaking incubator at 37°C and 180 rpm. This overnight culture was used as 1 % innocula into 4×1250 ml fresh medium and grown until the OD600 reached 0.6. The cultures were induced by addition of 10 ml/l of a filter sterilised 20 % w/v arabinose solution and growth continued at 37°C for two hours. Cells were harvested by centrifugation at 8000 rpm, 4°C, for 12 minutes and the cell paste, typically 20 g was stored at −80°C until required.
Dam was purified from 10 g of cell paste resuspended in 30 ml of buffer A [50 mM Tris/HCl (pH 9.0), 50 mM imidazole, 300 mM NaCl, 0.05 % v/v triton X-100, 10 % w/v glycerol, 10 mM 2-mercaptoethanol] and 0.3 ml of a 10 mg/ml lysozyme solution added. The suspension was stirred for 20 minutes at 4°C and then sonicated 25 times for 5 second bursts. The lysate was cleared by centrifugation at 12,000 rpm and 4°C for 30 min. The supernatant was applied to a nickel charged chelating sepharose FF column (5 ml bed volume) previously equilibrated in buffer A. The column was washed with 200 ml of buffer A and the proteins eluted with a 30 ml gradient to 100 % buffer B (buffer A plus 500 mM imidazole). The purest fractions (8 ml) were pooled and dialysed twice for 30 min at 4°C against 500 ml buffer C [50 mM Tris/HCl (pH 7.5), 200 mM NaCl, 0.2 mM EDTA, 20 % w/v glycerol, 2 mM dithiothreitol]. The purification yielded 1.5 mg Dam from 10 g cell paste and aliquots of protein solution (100 µl) were immediately frozen at −80°C.
Dam activity assay
Fluorescence changes were recorded in a Tecan Safire 2 microplate reader using 10 readings per well (each measurement), 0.5 s between each movement and reading, 1 second of shaking between data collections with 7 seconds of settle time. The following instrument settings were used: the excitation wavelength was 486 nm, the emission wavelength was 518 nm, the bandwidth was 5 nm, the gain was 200, the Z-position was 9300 µm and the integration time was 40 µs. Break light oligonucleotide sequences used in the assay were; oligonucleotide 1: 5′ C(F)CGGAmTCCAGTTTTCTGGATCCGG(D) 3′; oligonucleotide 2: 5′ C(F)CGGAmTCCAGTTTTCTGGAmTCCGG(D) 3′, where Dam recognition sequences are shown in bold, (F) represents fluorescein and (D) represents a dabcyl quencher.
The activity of Dam was measured in triplicate in Greiner flat bottomed black 96 well polypropylene microplates, with a total assay volume of 200 µl, maintained at 37°C. Three buffers were required: buffer D [22.4 mM Tris-acetate (pH 7.9), 23.5 mM sodium chloride, 56 mM potassium acetate, 11.2 mM magnesium acetate, 1.12 mM dithiothreitol, 0.12 mg/ml BSA] which could be modified by the addition of between 0 and 235 µM AdoMet and between 0 and 35 nM oligonucleotide 1; buffer E [20 mM Tris-acetate (pH 7.9), 50 mM potassium acetate, 10 mM magnesium acetate and 1 mM dithiothreitol]; buffer F [48 mM Tris/HCl (pH 7.4), 9.6 mM EDTA, 4.8 mM 2-mercaptoethanol, 0.4 mg/ml BSA and 30 nM oligonucleotide 1]. Dam solution was prepared by defrosting an aliquot of Dam stock (as purified, 0.17 mg/ml) on ice for 10 minutes and diluting it 510 fold into buffer F. DpnI solution was prepared by diluting the stock solution to 1 U/µl in buffer E.
170 µl of buffer D was added to each well of the plate and overlaid with 3 drops of mineral oil. The plate was then equilibrated at 37°C for 15 minutes and the reaction initiated by the addition of 10 µl of DpnI solution and 20 µl of Dam solution to each well. Fluorescein emission was then monitored over time. The rate of reaction was calculated by taking the initial rate of fluorescence change over 180 seconds unless otherwise stated. Background changes in fluorescence were accounted for by subtracting a negative control (lacking Dam) when appropriate. The rate of change in fluorescence was converted to a rate of reaction using a fluorescence calibration curve. Data were fitted with the program SigmaPlot.
Oligonucleotide 1 fluorescence calibration curve
Assays contained the fully methylated oligonucleotide 2 in place of 1 at concentrations 0, 0.5, 1, 2, 3, 3.5 nM, 25 µM AdoMet and no Dam. The endpoint fluorescence was measured.
Kinetic analysis of Y. pestis Dam
The dependence of enzyme activity on AdoMet concentration was measured in a series of assays containing 33 nM oligonucleotide 1, 1.0 nM Dam and 2.5, 5, 10, 20, 40, 100, 200 µM AdoMet. The dependence of reaction rate on oligonucleotide 1 concentration was measured in a series of assays containing 0.31 nM Dam, 120 µM AdoMet and 0.5, 1.5, 2.5, 3.5, 4.5, 5.5, 6.5 nM oligonucleotide 1.
Inactivation of Y. pestis Dam
The inactivation was monitored at 30°C under three conditions: in the absence of substrates, with 30 nM oligonucleotide 1 or with 120 µM AdoMet. The Dam solution was prepared by diluting Dam (as purified) 213 fold in buffer F lacking DNA. To 200 µl of Dam solution was added 100 µl of DpnI solution and the resultant mixture aliquotted into PCR tubes (35 µl in each). The PCR tubes were maintained at 30°C in a PCR machine and aliquots withdrawn at the required time points. Aliquots were then rapidly cooled in an ice bath and the activities assayed at the end of the timecourse.
Inhibition of Dam by S-adenosylhomocysteine
Reactions contained 1 nM Dam and 33 nM oligonucleotide 1, with varying AdoMet and S-adenosylhomocysteine concentrations: S-adenosylhomocysteine; 0, 5, 10, 15, 20, 30, 40 µM, AdoMet; 20, 25, 30, 40, 70, 140, 200 µM. KiSAH was estimated as follows: a double reciprocal plot () of 1/rate of reaction against 1/concentration of AdoMet has a slope of KM.app/Vmax, yielding a series of values for the apparent KM (KM.app) at different inhibitor concentrations. A plot of KM.app against concentration of S-adenosylhomocysteine () has intercepts of KM on the KM.app axis and –Ki on the concentration of S-adenosylhomocysteine axis.
High throughput screening validation conditions
The activity of Dam was measured in half area Greiner flat bottomed black 96 well polystyrene microplates, with a total assay volume of 100 µl, maintained at 30°C. Four buffers were required: buffer G [buffer D containing 5.9 µM AdoMet and 35 nM oligonucleotide 1 (final concentrations of 5 µM and 33 nM respectively in the assay)]; buffer H [buffer E containing 1 U/µl DpnI (final concentration 5 U per assay)]; buffer I [buffer F containing 20 nM Dam (for a final concentration of 2 nM)]. Per 96 well plate, 1160 µl buffer I was mixed with 580 µl buffer H to make buffer J.
Using a Biomek 3000 liquid handling system, 85 µl of buffer G was added to each well. The plate was then equilibrated at 30°C for 20 minutes and the reaction initiated by the addition of 15 µl of buffer J per well. The plate was then immediately transferred to a Tecan Saffire 2 microplate reader and fluorescein emission monitored over time. The following instrument settings were used: 10 readings per well (each measurement), 0 s between each movement and reading and no shaking between data collections. The excitation wavelength was 486 nm with a bandwidth of 9 nm, the emission wavelength was 518 nm with a bandwidth of 20 nm, the gain was 130, the Z-position was 9300 µm and the integration time was 40 µs. The rate of reaction was calculated by taking the initial rate of fluorescence change over the first 675 seconds. The rate of fluorescence increase was estimated by fitting data to a linear trend line with the program Excel.