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Recombinase Mechanisms

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  1. Recombinase Mechanisms

  2. Recombinase enzymes catalyze DNA insertion at specific attachment sites AttP : Phage attachment sites P O P’ O B O B’ O AttB : Bacterial attachment sites

  3. The DNA is integrated AttP : Phage attachment sites P O P’ O B O B’ O AttB : Bacterial attachment sites Integrase O B O P’ P O B’ 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 P O P’ O B O B’ O AttB : Bacterial attachment sites Integrase Integrase + Excisionase O B O P’ P O B’ O AttL AttR

  5. Re-arranging the recognition sites enables inversion rather than excision O P O P’ B’ O B O AttP AttB* Integrase Integrase + Excisionase O P O B’ P’ O B 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 EP SM 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 EP SM 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 EP SM 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),

  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 don’t 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 EP SM K-2 K2 LPM2 SM2 EMP2 SM4 K34 K3 K-3 K5 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. > 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. > 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 • Cooperativity (dimer binding > monomer) • Cre binds target 3x > than Flp

  24. DNA Binding [1] LP M Now we know Keq1 = K1/K-1 S Dissociation K1 K-1 EP SM K-2 K2 LPM2 SM2 EMP2 SM4 K34 K3 K-3 K5 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 EP SM K-2 K2 LPM2 SM2 EMP2 SM4 K34 K3 K-3 K5 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 • Cooperativity (dimer binding > monomer) • 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 EP SM 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. <10 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 < 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 - 3:1 optimum for Flp, 1:1 optimum for Cre

  40. 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

  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 - 3:1 optimum for Flp, 1:1 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 - 3:1 optimum for Flp, 1:1 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