recombinase mechanisms. recombinase enzymes catalyze dna insertion at specific attachment sites...
DESCRIPTION
OBB’O O AttB : Bacterial attachment sites P’BOO OP AttP : Phage attachment sites B’OPO Integrase AttLAttR The DNA is integratedTRANSCRIPT
Recombinase Mechanisms
Recombinase enzymes catalyze DNA insertion at specific attachment sites
OB B’O OAttB :
Bacterial attachment sites
OP P’
AttP :Phage attachment sites
OB B’O OAttB :
Bacterial attachment sites
P’BO O
OP P’
AttP :Phage attachment sites
B’OP O
Integrase
AttL AttR
The DNA is integrated
OB B’O OAttB :
Bacterial attachment sites
OP P’
AttP :Phage attachment sites
State is stable and directionality of reaction controlled by excisionase. So, it holds state and
switching is controllable.
Integrase
AttL AttRP’BO O B’OP O
Integrase +Excisionase
Re-arranging the recognition sites enables inversion rather than excision
Integrase
AttR AttL*P’ BO OB’OP O
Integrase +Excisionase
AttP AttB*P’ BO O B’ OP O
Cre, Flp inverted repeat target
Cre, Flp
Forward and reverse reactions
KN Equilibirum constant for conversion between complexes
.. that can be descried in cartoon form, just as the total
system can …
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S
SM
SM2
SM4
DNA binding to inverted repeat sites [1]
Synapsis [2] Recombination
Dissociation
[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).
IIEP
LP
EP
LPM2
EMP2
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S
SM
SM2
SM4
DNA Binding [1]
Synapsis [2] Recombination
Dissociation
[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).
IIEP
LP
EP
LPM2
EMP2
Parameters that describe system behavior within the
mechanistic model proposed can be defined.
M
S
SM
SM2
SM4
DNA Binding [1]
Synapsis [2] Recombination
[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),
K1
K2
K-1
K-2
K3
IIEP
LP
EP
LPM2
EMP2
K-4
K4
K-34
K34 K-5
Dissociation
K-3 K5
Parameters and model relationships provide basis for mathematical description of
the system. M
S
SM
SM2
SM4
K1
K2
K-1
K-2
But, we don’t know parameter values (association &
dissociation rate consts).
So, use assays to interrogate physical system and gather
data. Fit data to model to find parameters.
Data
Cartoon
Mathematical
Description
Parameters
CurveFitting &
Optimization
Set of parameters that describe recombination
system for Cre, Flp give us insights, such as :
Data
Cartoon
Mathematical
Description
CurveFitting &
Optimization
Parameters
Factors that drive recombination efficiency
M
S
SM
SM2
SM4
DNA Binding [1]
Synapsis [2] Recombination
[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),
K1
K2
K-1
K-2
K3
IIEP
LP
EP
LPM2
EMP2
K-4
K4
K-34
K34 K5
Dissociation
K-3 K-5
Start with measurement equilibrium
binding constants to
evaluate strength of binding and degree of
cooperativity
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.
Log of the molar concentration
Normal binding siteMolar concentration
Dimerization is dominant state as the concentration of recombinse
increases.
Log of the molar concentration
Normal binding siteMolar concentration
Theoretical [1] equilibrium distribution of DNA target between
three states (free, monomer & dimer bound) given by:
[1] Discussed in materials and methods
Fit data to equations to get equilibrium
constants for DNA bindingData
Model
Fitting
K1, K2
Equilibrium constants found for monomer [1] and dimer [2]
[1] For recombinase binding to single target site; check method used[2] As explained
Dimer binding much stronger than monomer binding, suggesting
cooperativity.
[1] For recombinase binding to single target site; check method used[2] As explained
~ 40x > 100x
Cooperativity characterized by decreased intermediates. This is seen here, with minimal
monomer intermediate present.Free
Monomer
Dimer
Cre binds target site with ~3x cooperativity relative to Flp.
[1] For recombinase binding to single target site; check method used[2] As explained
~ 40x > 100x
Found equilibrium binding constants using combination of mathematical model and
data. Learned : Data
Cartoon
Mathematical
Description
CurveFitting &
Optimization
Parameters
1. Cooperativity (dimer binding > monomer)
2. Cre binds target 3x > than Flp
M
S
SM
SM2
SM4
DNA Binding [1]
Synapsis [2] Recombination
[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),
K1
K2
K-1
K-2
K3
IIEP
LP
EP
LPM2
EMP2
K-4
K4
K-34
K34 K5
Dissociation
K-3 K-5
Now we know Keq1 = K1/K-1
M
S
SM
SM2
SM4
DNA Binding [1]
Synapsis [2] Recombination
[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),
K1
K2
K-1
K-2
K3
IIEP
LP
EP
LPM2
EMP2
K-4
K4
K-34
K34 K5
Dissociation
K-3 K-5
Next, with kinetic assays
findK1 and K-1
Monomer present at earl time points, replaced by dimer complex.
FLP Cre
Cre is faster.
FLP Cre
Dynamic model to simulate the timecourse of DNA binding without parameters.
Fit [1] model to data to find parameters
Data
Model
Fitting
…
[1] Use optimization procedure.
Get a set of association and dissociation rate constants
across the recombinase concentrations.
[1] Nearly identical across protein concentraions[2] Macroscopic association rate constants
Dissociation rate for dimer (K-2) is 10x less than for monomer (K-1),
suggesting again cooperativity in binding.
Higher binding affinity for Cre : faster association rate and smaller
dissociation of the dimer.
Found association and dissociation rate constant for Cre, Flp using combination of
mathematical model and data. Data
Cartoon
Mathematical
Description
CurveFitting &
Optimization
Parameters
1. Cooperativity (dimer binding > monomer)2. Cre binds stronger: dimer has faster
association rate and slower dissocation rate than Flp
M
S
SM
SM2
SM4
DNA Binding [1]
Synapsis [2] Recombination
[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),
K1
K2
K-1
K-2
K3
IIEP
LP
EP
LPM2
EMP2
K-4
K4
K-34
K34
Dissociation
K-3
Now that DNA binding is described, find parameters that
describe recombination and use
to gain insights.K-5 K5
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.
[1] Normalized substrate at 0.4 nM, 60 minute reaction
~20nM ~2nM
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
[1] 0.4 nM substrate; timecourse at optimal concentrations : 25.6 nM FLP and 2.4 nM Cre b b
<10 minutes needed to approach maximum excision for both at optimal substrate
concentration..
[1] 0.4 nM substrate; timecourse at optimal concentrations : 25.6 nM FLP and 2.4 nM Cre
Cre excision limited at < 75%. Investigated further with substrate titration.
Substrate titration reveals more features.
[1] 0.4 nM substrate (25.6 nM FLP and 2.4 nM Cre). Open: 3 min, closed 60 min[2] 1/5 - 3:1 optimum for Flp, 1:1 optimum for Cre
60 mins
3 mins
Sharp reduction when binding sites > 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 - 3:1 optimum for Flp, 1:1 optimum for Cre
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 - 3:1 optimum for Flp, 1:1 optimum for Cre
[1] 0.4 nM substrate (25.6 nM FLP and 2.4 nM Cre). Open: 3 min, closed 60 min[2] 1/5 - 3:1 optimum for Flp, 1:1 optimum for Cre
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?
Mathematical model used to determine parameters responsible for behavior of Cre, Flp and investigate
Cre excision rate.Substrate titration
data
Model (13 ODEs)
Fitting & optimization
K34, K-34, K5, K-5
DNA binding affinityRate constants
(previously determined)
Get set of optimized parameters.
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
Model predicts that the 50 to 75% maximum level of excision for Crereflects an equilibrium between
excision and integration, which is due to the high stability of
the synaptic complex.
Punchline.
IEP
K-34 K5
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Drivers of recombination inefficiency:
1. Low-affinity DNA-monomer binding
IEP
K-34 K5
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Drivers of recombination inefficiency:
1. Low-affinity DNA-monomer binding
2. Synaptic stability
IEP
K-34 K5
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Story of Flp: Low-affinity DNA-monomer
bindingrequiring 10x more protein
than Cre for DNA binding, yet also achieving 100%
recombination.
IEP
K-34 K5
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Story of Cre: High-affinity DNA-monomer
bindingrequiring 10x less protein than
Flp, yet achieving <75% recombination due to synaptic
stability.
IEP
K-34 K5
M
S I
Punchline. Likely an optimum that
balance DNA binding affinity and synaptic stability.
Punchline. Parameters and mechanistic model establish a basis for
incorporating recombination in dynamic model for counter
architecture.