Recombinase Mechanisms

1
Recombinase Mechanisms
2
Recombinase enzymes catalyze DNA insertion at
specific attachment sites
AttP Phage attachment sites
O
P
P
O
B
B
O
O
AttB Bacterial attachment sites
3
The DNA is integrated
AttP Phage attachment sites
O
P
P
O
B
B
O
O
AttB Bacterial attachment sites
Integrase
P
B
O
O
B
O
P
O
AttL
AttR
4
State is stable and directionality of reaction
controlled by excisionase. So, it holds state and
switching is controllable.
AttP Phage attachment sites
O
P
P
O
B
B
O
O
AttB Bacterial attachment sites
Integrase
Integrase Excisionase
P
B
O
O
B
O
P
O
AttL
AttR
5
Re-arranging the recognition sites enables
inversion rather than excision
P
B
O
O
B
O
P
O
AttP
AttB
Integrase
Integrase Excisionase
P
B
O
O
B
O
P
O
AttR
AttL
6
.. that can be descried in cartoon form, just as
the total system can
KN
Equilibirum constant for conversion between
complexes
Forward and reverse reactions
Cre, Flp
Cre, Flp inverted repeat target
7
DNA binding to inverted repeat sites 1
LP
M
S
Dissociation
SM
EP
LPM2
SM2
EMP2
SM4
Recombination
Synapsis 2
I
IEP
1 Bind as monomer, then form a dimer upon
second monomer binding. (Andrews et al., 1987
Hoess et al., 1984 Mack et al., 1992). 2 FLP
synapsis occurs by random collision (Beatty et
al., 1986). For Cre, synapsis in vitro occurs by
random collision, but may be achieved by an
ordered mechanism (Adams et al., 1992).
8
DNA Binding 1
LP
M
S
Dissociation
SM
EP
Parameters that describe system behavior within
the mechanistic model proposed can be defined.
LPM2
SM2
EMP2
SM4
Recombination
Synapsis 2
I
IEP
1 Bind as monomer, then form a dimer upon
second monomer binding. (Andrews et al., 1987
Hoess et al., 1984 Mack et al., 1992). 2 FLP
synapsis occurs by random collision (Beatty et
al., 1986). For Cre, synapsis in vitro occurs by
random collision, but may be achieved by an
ordered mechanism (Adams et al., 1992).
9
DNA Binding 1
LP
M
S
Dissociation
K1
K-1
SM
EP
K-2
K2
LPM2
SM2
EMP2
SM4
K34
K3
K-5
K-3
K5
K-34
Recombination
Synapsis 2
K4
I
IEP
K-4
1 Bind as monomer, then form a dimer upon
second monomer binding. (Andrews et al., 1987
Hoess et al., 1984 Mack et al., 1992). 2(for
reviews, see Stark et al., 1992 Jayaram, 1994
Sadowski, 1995),
10
Parameters and model relationships provide basis
for mathematical description of the system.
M
S
K1
K-1
SM
K-2
K2
SM2
SM4
11
But, we dont know parameter values (association
dissociation rate consts).
12
So, use assays to interrogate physical system and
gather data. Fit data to model to find parameters.
Data
Curve Fitting Optimization
Parameters
Mathematical Description
Cartoon
13
Set of parameters that describe recombination
system for Cre, Flp give us insights, such as
Data
Curve Fitting Optimization
Parameters
Mathematical Description
Cartoon
Factors that drive recombination efficiency
14
DNA Binding 1
LP
M
Start with measurement equilibrium binding
constants to evaluate strength of binding and
degree of cooperativity
S
Dissociation
K1
K-1
SM
EP
K-2
K2
LPM2
SM2
EMP2
SM4
K34
K3
K5
K-3
K-5
K-34
Recombination
Synapsis 2
K4
I
IEP
K-4
1 Bind as monomer, then form a dimer upon
second monomer binding. (Andrews et al., 1987
Hoess et al., 1984 Mack et al., 1992). 2(for
reviews, see Stark et al., 1992 Jayaram, 1994
Sadowski, 1995),
15
Mobility shift data measures distribution of DNA
target between three states (free, bound to Flp
monomer Flp dimer bound) with respect to
increasing Flp concentration.
Normal binding site
Molar concentration
Log of the molar concentration
16
Dimerization is dominant state as the
concentration of recombinse increases.
Normal binding site
Molar concentration
Log of the molar concentration
17
Theoretical 1 equilibrium distribution of DNA
target between three states (free, monomer
dimer bound) given by
1 Discussed in materials and methods
18
Fit data to equations to get equilibrium
constants for DNA binding
Fitting
Data
Model
K1, K2
19
Equilibrium constants found for monomer 1 and
dimer 2
1 For recombinase binding to single target
site check method used 2 As explained
20
Dimer binding much stronger than monomer binding,
suggesting cooperativity.
gt 100x
40x
1 For recombinase binding to single target
site check method used 2 As explained
21
Cooperativity characterized by decreased
intermediates. This is seen here, with minimal
monomer intermediate present.
Free
Dimer
Monomer
22
Cre binds target site with 3x cooperativity
relative to Flp.
gt 100x
40x
1 For recombinase binding to single target
site check method used 2 As explained
23
Found equilibrium binding constants using
combination of mathematical model and data.
Learned
Data
Curve Fitting Optimization
Parameters
Mathematical Description
Cartoon

1. Cooperativity (dimer binding gt monomer)
2. Cre binds target 3x gt than Flp

24
DNA Binding 1
LP
M
Now we know Keq1 K1/K-1
S
Dissociation
K1
K-1
SM
EP
K-2
K2
LPM2
SM2
EMP2
SM4
K34
K3
K5
K-3
K-5
K-34
Recombination
Synapsis 2
K4
I
IEP
K-4
1 Bind as monomer, then form a dimer upon
second monomer binding. (Andrews et al., 1987
Hoess et al., 1984 Mack et al., 1992). 2(for
reviews, see Stark et al., 1992 Jayaram, 1994
Sadowski, 1995),
25
DNA Binding 1
LP
M
Next, with kinetic assays find K1 and K-1
S
Dissociation
K1
K-1
SM
EP
K-2
K2
LPM2
SM2
EMP2
SM4
K34
K3
K5
K-3
K-5
K-34
Recombination
Synapsis 2
K4
I
IEP
K-4
1 Bind as monomer, then form a dimer upon
second monomer binding. (Andrews et al., 1987
Hoess et al., 1984 Mack et al., 1992). 2(for
reviews, see Stark et al., 1992 Jayaram, 1994
Sadowski, 1995),
26
Monomer present at earl time points, replaced by
dimer complex.
Cre
FLP
27
Cre is faster.
Cre
FLP
28
Dynamic model to simulate the timecourse of DNA
binding without parameters.
29
Fit 1 model to data to find parameters
Fitting
Data
Model

1 Use optimization procedure.
30
Get a set of association and dissociation rate
constants across the recombinase concentrations.
1 Nearly identical across protein
concentraions 2 Macroscopic association rate
constants
31
Dissociation rate for dimer (K-2) is 10x less
than for monomer (K-1), suggesting again
cooperativity in binding.
32
Higher binding affinity for Cre faster
association rate and smaller dissociation of the
dimer.
33
Found association and dissociation rate constant
for Cre, Flp using combination of mathematical
model and data.
Data
Curve Fitting Optimization
Parameters
Mathematical Description
Cartoon

1. Cooperativity (dimer binding gt monomer)
2. Cre binds stronger dimer has faster association
   rate and slower dissocation rate than Flp

34
DNA Binding 1
LP
M
Now that DNA binding is described, find
parameters that describe recombination and use to
gain insights.
S
Dissociation
K1
K-1
SM
EP
K-2
K2
LPM2
SM2
EMP2
SM4
K34
K3
K-3
K-5
K5
K-34
Recombination
Synapsis 2
K4
I
IEP
K-4
1 Bind as monomer, then form a dimer upon
second monomer binding. (Andrews et al., 1987
Hoess et al., 1984 Mack et al., 1992). 2(for
reviews, see Stark et al., 1992 Jayaram, 1994
Sadowski, 1995),
35
In vitro recombination assay 10x more Flp
required to reach maximum excision of a given
quantity of substrate than Cre. This is due to
the fact that Cre has higher binding affinity.
20nM
2nM
1 Normalized substrate at 0.4 nM, 60 minute
reaction
36
Enzymes required in excess over substrate for
efficient recombination. Makes sense because this
is not 1 enzyme, 1 substrate class for excision
all four binding sites must be occupied
simultaneously for long enough for synapsis.
1 Normalized substrate at 0.4 nM, 60 minutes
37
lt10 minutes needed to approach maximum excision
for both at optimal substrate concentration..
b
b
1 0.4 nM substrate timecourse at optimal
concentrations 25.6 nM FLP and 2.4 nM Cre
38
Cre excision limited at lt 75. Investigated
further with substrate titration.
1 0.4 nM substrate timecourse at optimal
concentrations 25.6 nM FLP and 2.4 nM Cre
39
Substrate titration reveals more features.
60 mins
3 mins
1 0.4 nM substrate (25.6 nM FLP and 2.4 nM
Cre). Open 3 min, closed 60 min 2 1/5 - 31
optimum for Flp, 11 optimum for Cre
40
Sharp reduction when binding sites gt Cre monomer,
yet no analogous reduction seen for Flp. Higher
binding affinity of Cre results in exhaustion of
monomers when substrate saturated.
1 0.4 nM substrate (25.6 nM FLP and 2.4 nM
Cre). Open 3 min, closed 60 min 2 1/5 - 31
optimum for Flp, 11 optimum for Cre
41
Flp recombines 100 of substrate across wide
range of concentrations. Lower Flp binding
affinity lets it recombine high fraction of
substrate even when substrate is in excess.
1 0.4 nM substrate (25.6 nM FLP and 2.4 nM
Cre). Open 3 min, closed 60 min 2 1/5 - 31
optimum for Flp, 11 optimum for Cre
42
Cre does not exceed 75 excision even when
protein in excess. Why? Recombination sharply
reduced when number of sites exceeds monomers due
to what? Higher binding affinity (cooperativity),
protein aggregation, non-specific binding?
1 0.4 nM substrate (25.6 nM FLP and 2.4 nM
Cre). Open 3 min, closed 60 min 2 1/5 - 31
optimum for Flp, 11 optimum for Cre
43
Mathematical model used to determine parameters
responsible for behavior of Cre, Flp and
investigate Cre excision rate.
Fitting optimization
Substrate titration data
DNA binding affinity Rate constants (previously
determined)
K34, K-34, K5, K-5
Model (13 ODEs)
44
Get set of optimized parameters.
45
k5, corresponding to the dissociation of the
recombined synapse, is approximately 30-fold
larger for FLP than for Cre. K-5, describing the
reassociation of protein bound recombination
products into the synaptic complex, is
approximately tenfold larger for Cre than for FLP
46
Model predicts that the 50 to 75 maximum level
of excision for Cre reflects an equilibrium
between excision and integration, which is due to
the high stability of the synaptic complex.
47
Punchline.
48
Drivers of recombination inefficiency 1.
Low-affinity DNA-monomer binding
M
K-34
K5
IEP
I
S
49
Drivers of recombination inefficiency 1.
Low-affinity DNA-monomer binding 2. Synaptic
stability
M
K-34
K5
IEP
I
S
50
Story of Flp Low-affinity DNA-monomer
binding requiring 10x more protein than Cre for
DNA binding, yet also achieving 100
recombination.
M
K-34
K5
IEP
I
S
51
Story of Cre High-affinity DNA-monomer
binding requiring 10x less protein than Flp, yet
achieving lt75 recombination due to synaptic
stability.
M
K-34
K5
IEP
I
S
52
Punchline. Likely an optimum that balance DNA
binding affinity and synaptic stability.
M
K-34
K5
IEP
I
S
53
Punchline. Parameters and mechanistic model
establish a basis for incorporating recombination
in dynamic model for counter architecture.