In vitro DNA binding, unwinding and glycosylase assays
DNA strands for substrate formation (supplemental data) were synthesized with Cy5 end-label. DNA binding was assessed using electrophoretic mobility shift assays (EMSAs). Reactions were incubated at 37°C for 20 minutes in helicase buffer (HB); 20 mM Tris pH 7.5, 10% (v/v) glycerol, 100 µg/ml BSA, using 12.5 nM Cy5-fluorescently labelled DNA substrate, 25 mM DTT and 5 mM EDTA, and then placed on to ice for 10 minutes. Orange G and 80% (v/v) glycerol (OG) was added to load reactions onto a 5% acrylamide TBE gel that was electrophoresed for 1 hour 30 minutes at 140 V. Gels were imaged using a Typhoon phosphor-imager (Amersham) at 633 nm using a R765 filter for Cy5 detection.
DNA unwinding assays were at 37°C in reactions containing buffer HB, 12.5 nM Cy5-fluorescently labelled DNA substrate, 25 mM DTT, 1.25 µM unlabeled ‘trap’ DNA, 5 mM ATP and 5 mM CaCl2. Reactions were pre-incubated at 37°C for 5 minutes without the ‘trap’ or ATP before they were added together to start the reactions for 30 minutes at 37°C, stopped by addition of stock stop solution (4 µl per 20 µl reaction); 2 mg/mL proteinase K in 200 mM EDTA and 2.5% (w/v) SDS. OG dye was added for electrophoresis through a 10% acrylamide TBE gel for 45 minutes at 150 V. Gels were imaged using a Typhoon phosphor-imager (Amersham) at 633 nm using a R765 filter for Cy5 detection.
DNA glycosylase reactions were at 37°C in reaction mixtures containing buffer HB, 12.5 nM Cy5-fluorescently labelled DNA substrate, 25 mM DTT, 5 mM ATP, 4 mM MnCl2 and 4 mM CaCl2. Reactions were pre-incubated at 37°C before being initiated by addition of Lhr protein and (unless in a time course assay) allowed to continue for 30 minutes before addition of stock stop solution and 4 µl of 1 M NaOH. Reaction samples were boiled for 5 minutes and formamide added before loading into a 15% denaturing (8 M urea) acrylamide TBE gel for 4 hours at 5 watts per gel. Gels were imaged using a Typhoon phosphor-imager (Amersham) at 633 nm using a R765 filter for Cy5 detection, generating TIFFs that were measured using Gel Analyzer 19.1 (Lazar) software. Graphs of glycosylase activity were generated using Prism (GraphPad).

Generation of a chromosomal deletion ofE. coli lhr

DNA constructs and strain genotypes are presented in the Supplementary material. Lhr deletion was by recombineering [24] and P1 transduction of an FRT (FLP recognition target) flanked Kanr marker. To generate an effective P1 stock the overnight culture was used to inoculate 8 ml of Mu broth containing 6 mM CaCl2. A sample of the cells (0.1 mL) grown at 37°C to OD600 0.8-1.0 in a shaking water bath was added to four overlay tubes each containing 3 mL of 0.4% w/v Mu broth agar held at 42°C. P1 phage stock was diluted 10-100-fold in MC buffer (100 mM MgSO4, 5 mM CaCl2) and 0.05 mL, 0.1 mL or 0.2 mL of this diluted phage was added to the overlay tubes containing cells and molten agar and gently mixed. The remaining tube was left without phage as a control. The contents of each overlay tube was poured onto P1 agar plates and left to set for overnight growth at 37°C for 18 hours. Soft agar from phage-lysed plates was added to 1 ml of MC buffer (100 mM MgSO4 and 5 mM CaCl2) and 0.5 ml of chloroform for vigorous mixing before centrifugation at 5752 rcf for 20 minutes at 4°C. The supernatant was retrieved and mixed with chloroform (0.5 mL) for storage at 4°C as a P1 phage stock. MG1655 recipient strain was grown in a Mu Broth to OD600 0.8 using a shaking water bath. Cells were pelleted, resuspended in 1 ml of MC buffer, and left at 25°C for 10 minutes. 0.2 ml of cells were added into 3 overlay tubes containing 0 ml, 0.05 ml and 0.2 ml of P1 lysate produced previously and incubated for 30 minutes at 37°C. Cell/P1 lysate mix was added to 2.5 ml of 0.6% agar, mixed gently and poured onto Mu Broth agar plates containing 30 µg/ml kanamycin and left to set. Plates were grown for 1-2 days, lid-up, at 37°C to allow colonies to develop. Colonies were then picked and purified by streaking onto Mu broth agar plates containing no antibiotic. This was repeated 3 times before plating again onto agar containing 30 µg/ml kanamycin for confirmation of gene knockout and Kanr-FTR insertion.
Successful P1 treated MG1655 cells were transformed with pCP20. Transformants were picked and used to inoculate 8 ml of Mu broth containing no antibiotic. Culture was grown overnight at 45°C in a shaking water bath FLP recombinase expression and plasmid curation. Cells were then streaked onto Mu Broth agar plates to produce single colonies and grown at 37°C overnight. Colonies were re-streaked 3 times before replica plating onto Mu Broth agar plates containing 50 µg/ml ampicillin, 30 µg/ml kanamycin and then no antibiotic to confirm loss of the pCP20 plasmid. Isolates which only grew on the no antibiotic agar plates were grown overnight for glycerol stock production and streaked a further time for colony PCR diagnostic confirmation.

E. coli viability spot assays

Cell viabilities were measured from liquid cultures grown to OD600 0.3-0.4 in a shaking water bath at 37°C monitored in the growth tubes by using a Spectronic 20+. Cells were then treated by addition to the growth media of hydrogen peroxide (H2O2) or AZT at concentrations stated in the results. Cells were grown for a further 30 minutes and then serially diluted into 1x M9 medium to arrest growth for spotting (10 ul) on to agar plates grown overnight in a 37°C incubator. For comparing growth curves cells were grown to OD600 0.3-0.4 and then transferred into a 24-well flat-bottomed plate and H2O2 was added to appropriate wells to the given concentration from a 0.98 M stock. Growth in the plates was monitored with orbital shaking in a FLUOstar microplate reader (BMG Labtech). OD600 readings were taken every 30 minutes in this time, and data was extracted and analyzed using Prism (GraphPad) software.