RecA-mediated LexA hydrolysis kinetics
RecA-filament reaction: 10 M RecA, 2.2 M oligo (18-mer), 1 mM ATP-γ-S, 0.25X
NEB RecA reaction buffer. Follow order of addition:
58.6 l
H2O
2.5 l
10X NEB RecA reaction buffer
10 l
22 M SKBT25 oligo
10 l
10 mM ATP-γ-S
18.9 l
NEB RecA 2 mg/ml, 52.85 M
Inc O/N on ice
1.
2.
3.
Calculate or use xl spreadsheet to determine reaction conditions for 8 points of
[LexA] from ¼ Km to 5 Km (for wt LexA, this is ~0.2-4.5 M)
Place 96-well PCR plate on top of 30 oC heat block
Array 100 l reactions (without RecA filaments), RecA filaments and 10 l 2X
SDS-PAGE sample buffer in each “time” well as follows:
A
B
0.2
0.4
[LexA] M
10 10
RecA l
Time
0” 0”
Time
15”
Time
30”
Time
1’
1’
Time
1.5’
Time
2’
Time
Time
Time
Time
4.
5.
6.
7.
8.
9.
C
0.6
10
0”
1.5’
2’
3’
D
0.8
10
0”
2’
3’
4’
E
1.2
10
0”
4’
6’
8’
F
G
H
1.6 2.4 4.5
10 10 10
0” 0” 0”
12’ 12’ 12’
16’ 16’
24’
6’
8’
8’
1
2
3
4
5
6
7
8
9
10
11
12
After removing 10 l for 0” time point, start reaction by transferring 5 l RecA
filaments to 1st row using an 8-channel pipet.
Move 10 l from reaction wells to “Time” wells at designated time point
Seal plate and inc 95 oC 10’ using PCR machine.
Load 60 ng LexA for each time point onto 15% SDS-PAGE gel and silver stain
Determine Vi = |d [LexA] / d time| * [LexA]i by plotting in excel, determining
absolute slope with linear fit and multiplying by initial [LexA]
Fitting to Michaelus-Menton curve using GNU plot:
a. Place data in excel wherein 1st column is [LexA], 2nd column is Vi
b. Save .xls file as txt and rename to lexa.dat
c. Open GNUPlot
i. gnuplot> plot ‘C:\Documents… lexa.dat’ u 1:2
ii. # “u 1:2” means using columns 1 and 2
iii. gnuplot> f(x)=a*x/(b+x)
iv.
v.
vi.
vii.
gnuplot> a = 1
gnuplot> b = 1.5
gnuplot> fit f(x) ‘C:\Documents… lexa.dat’ via a,b
# This fits the data to MM curve and returns a=Vmax and b=Km
Notes:
Reported [RecA-filaments] varies from 20 nM (Geise and Little JMB08) to 1 uM
(Takahashi and Schnarr EuJBiochem89). Activation reaction:
Range of [substrate]’s should be from ¼ Km to 5 Km. For wt LexA, this is ~0.2-4.5 uM.
From Geise and Little JMB08:
LexA cleavage
LexA autodigestion was performed in 100 mM Ncyclohexyl-3-aminopropane sulfonic acid–NaOH, pH 10.4, and 200
mM NaCl at 37 °C. RecA-dependent cleavage of LexA was performed as previously described,47 with the following
modifications: RecA was activated with the oligo SKBT25 (GCG TGT GTG GTG GTG TGC), which was selected for
preferential binding to RecA.24 RecA activation reactions typically contained 10 μM RecA, 2.2 μM oligo (or 40 μM
nucleotides), 20 mM Tris, pH 7.4, 2 mM MgCl2, 1 mM ATP-γ-S, and 1 mM DTT and were incubated overnight on ice.
RecA activity was constant between 3 and 5 nucleotides per RecA molecule (data not shown); a 1:4 ratio was used. The
long activation time produced higher activity than incubations of 2 h or less (data not shown), possibly due to slow
repacking of RecA to the oligo to eliminate gaps between RecA molecules. Cleavage reactions were performed in 20
mM Tris, pH 7.4, 5 mMMgCl2, 1 mM ATP-γ-S, 1 mM DTT, 2 mM Pipes, 40 mM NaCl, 0.02 mM EDTA, and 2%
glycerol. The last four components came from the addition of LexA and LexA storage buffer to make up 20% of the
volume of all reactions. This afforded constant ionic conditions, even when large volumes of LexA were used. Unless
otherwise indicated, cleavage was initiated by adding RecA to prewarmed samples. Aliquots were stopped at various
times by adding sample buffer to a final concentration of 30 mM Tris, pH 6.8, 5% glycerol, 2% SDS, 0.01%
bromphenol blue, and 2.5% β-mercaptoethanol. Samples were run on 15% Laemmli gels50 and stained with Coomassie
blue.