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.