recombinase mechanisms. recombinase enzymes catalyze dna insertion at specific attachment sites...

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 …

M

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

M

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

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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.