In vitro RecA-mediated LexA hydrolysis
RecA-filament reaction: 17.5 mM Tris-HCl pH 7.6, 2.5 mM MgCl2, 1.25 mM dTT
(0.25X NEB RecA reaction buffer), 1 mM ATP-γ-S, 10 M RecA, 2.2 M oligo (18mer). Follow order of addition into PCR tube:
8.79 l
H2O q.s. 15 l
0.375 l
10X NEB RecA reaction buffer
1.5 l
22 M SKBT25 oligo
1.5 l
10 mM ATP-γ-S
2.84 l
NEB RecA 2 mg/ml, 52.85 M
15 l
Inc 2 hr- O/N on ice
1. Place PCR plate in 30 oC water bath.
2. Array tubes with 2.5 l 5X SDS-PAGE sample buffer for each “time” point as
follows: 0.5’ 1’ 2’ 3’ 4’ 6’ 8’ 12’ 20’ 36’ or similar to fill 10 wells below RecA*
and LexA wells.
3. RecA-LexA cleavage reaction: x M LexA, 100 nM RecA*. 10 time points
possible with 10 l samples. Follow order of addition into above PCR tube:
Master Mix for Row 1:
0.25X NEB RecA reaction buffer
10X BSA (1 mg/ml)
RecA* from above
Array 85 l for each well in Row 1
per well
72.9 l
11 l
1.1 l
Master Mix x9
656 l
99 l
9.9 l
Individual for each well Row 2:
LexA storage buffer
q.s. each well 25 l
LexA to achieve 0.2, 0.4, 0.8, 1.4, 2, 3, 4, 8 M [final] in reaction
Equilibrate plate at 30 oC 5’. To start reaction add pre-mixed LexA from Row 2 to RecA*
in Row 1 using 8-channel pipete.
Map of 96-well plate:
RecA l
LexA M
Time
Time
Time
Time
Time
Time
Time
Time
Time
Time
5.
6.
7.
8.
9.
A
B
C
D
E
F
G
H
85
85
85
85
85
85
85
85
0.2 0.4 0.8 1.4
2
3
4
8
0.5” 0.5” 0.5” 0.5” 0.5” 0.5” 0.5” 0.5”
1’
1’
1’
1’
1’ 1’
1’
1’
2’
2’
2’
2’
2’
2’
2’
2’
3’
3’
3’
3’
3’
3’
3’
3’
4’
4’
4’
4’
4’
4’
4’
4’
6’
6’
6’
6’
6’
6’
6’
6’
8’
8’
8’
8’
8’
8’
8’
8’
12’ 12’ 12’ 12’ 12’ 12’ 12’ 12’
20’ 20’ 20’ 20’ 20’ 20’ 20’ 20’
36’ 36’ 36’ 36’ 36’ 36’ 36’ 36’
1
2
3
4
5
6
7
8
9
10
11
12
4. Remove 10 l from reaction and place in each time point’s designated well: 0.5,
1, 2, 3, 4, 6, 8, 12, 20, 36’.
Seal with PCR plate lid and inc 95 oC 10’ in PCR machine. Quick spin plate if
condensation on lid is a problem.
Load 10 l of 1:100 diluted protein ladder and each time point onto 15% SDSPAGE gel; SYPRO orange stain.
On GE Typhoon, try 450, 500 & 550 PMT to get max signal without saturation in
LexA bands. Note best data image as saturation not detected in ImageJ.
Determine band intensities using LukeMiller.org method for both full-length
LexA and fragment. With SYPRO orange, have dynamic range ~3 - 800 ng.
Determine Vi = |d [LexA] / d time| * [LexA]i by plotting in excel, determining
absolute slope with linear fit and multiplying by initial [LexA]
Notes:
Reported [RecA-filaments] varies from 20 nM (Geise and Little JMB08) to 1 uM
(Takahashi and Schnarr EuJBiochem89). Activation reaction can be incubated from 2 hr
to overnight on ice.
Loss of full-length LexA without increase of LexA fragment band intensity over time
may indicate aggregation of LexA.
May need to scout out optimum [RecA*] in 2-fold increments (e.g. try 50, 100, 200 nM
RecA* with [LexA] at ~1/4 Km and 5 Km).
Activation reaction:
Activation of the RecA filament (10 μM), carried out on ice for 2 h, and the RecA*-induced (2 μM)
cleavage of LexA (1.8 μM) at 37o C interacting with specific or non-specific DNA (~1.5 μM) were
performed as described previously for the unbound LexA repressor (16). (Butala, Zgur-Bertok NAR11)
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. (From Geise and Little JMB08)