1. Recombinase Mechanisms

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

3. OBB’O O AttB : Bacterial attachment sites P’BOO OP AttP : Phage attachment sites B’OPO Integrase AttLAttR The DNA is integrated

4. OBB’O O AttB : Bacterial attachment sites OPP’ AttP : Phage attachment sites State is stable and directionality of reaction controlled by excisionase. So, it holds state and switching is controllable. Integrase AttLAttR P’BOOB’OPO Integrase + Excisionase

5. Re-arranging the recognition sites enables inversion rather than excision Integrase AttRAttL* P’BOOB’OPO Integrase + Excisionase AttPAttB* P’BOOB’OPO

6. Cre, Flp inverted repeat target Cre, Flp Forward and reverse reactions KNKN Equilibirum constant for conversion between complexes.. that can be descried in cartoon form, just as the total system can …

7. 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). I IEP LP EP LPM2 EMP2

8. 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). I IEP LP EP LPM2 EMP2 Parameters that describe system behavior within the mechanistic model proposed can be defined.

9. 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), K1K1 K2K2 K -1 K -2 K3K3 I IEP LP EP LPM2 EMP2 K -4 K4K4 K -34 K 34 K -5 Dissociation K -3 K5K5

10. Parameters and model relationships provide basis for mathematical description of the system. M S SM SM2 SM4 K1K1 K2K2 K -1 K -2

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 Cartoon Mathematical Description Parameters Curve Fitting & Optimization

13. Set of parameters that describe recombination system for Cre, Flp give us insights, such as : Data Cartoon Mathematical Description Curve Fitting & Optimization Parameters Factors that drive recombination efficiency

14. 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), K1K1 K2K2 K -1 K -2 K3K3 I IEP LP EP LPM2 EMP2 K -4 K4K4 K -34 K 34 K5K5 Dissociation K -3 K -5 Start with measurement equilibrium binding constants to evaluate strength of binding and degree of cooperativity

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. Log of the molar concentration Normal binding site Molar concentration

16. Dimerization is dominant state as the concentration of recombinse increases. Log of the molar concentration Normal binding site 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 Data Model Fitting 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. [1] For recombinase binding to single target site; check method used [2] As explained ~ 40x > 100x

21. Cooperativity characterized by decreased intermediates. This is seen here, with minimal monomer intermediate present. Free Monomer Dimer

22. 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

23. Found equilibrium binding constants using combination of mathematical model and data. Learned : Data Cartoon Mathematical Description Curve Fitting & Optimization Parameters 1.Cooperativity (dimer binding > monomer) 2.Cre binds target 3x > than Flp

24. 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), K1K1 K2K2 K -1 K -2 K3K3 I IEP LP EP LPM2 EMP2 K -4 K4K4 K -34 K 34 K5K5 Dissociation K -3 K -5 Now we know K eq1 = K 1 /K -1

25. 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), K1K1 K2K2 K -1 K -2 K3K3 I IEP LP EP LPM2 EMP2 K -4 K4K4 K -34 K 34 K5K5 Dissociation K -3 K -5 Next, with kinetic assays find K 1 and K -1

26. Monomer present at earl time points, replaced by dimer complex. FLP Cre

27. Cre is faster. FLP Cre

28. Dynamic model to simulate the timecourse of DNA binding without parameters.

29. Fit [1] model to data to find parameters Data Model Fitting … [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 Cartoon Mathematical Description Curve Fitting & Optimization Parameters 1.Cooperativity (dimer binding > monomer) 2.Cre binds stronger: dimer has faster association rate and slower dissocation rate than Flp

34. 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), K1K1 K2K2 K -1 K -2 K3K3 I IEP LP EP LPM2 EMP2 K -4 K4K4 K -34 K 34 Dissociation K -3 Now that DNA binding is described, find parameters that describe recombination and use to gain insights. K -5 K5K5

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. [1] Normalized substrate at 0.4 nM, 60 minute reaction ~20nM~2nM

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. [1] 0.4 nM substrate; timecourse at optimal concentrations : 25.6 nM FLP and 2.4 nM Cre bbbb <10 minutes needed to approach maximum excision for both at optimal substrate concentration..

38. [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.

39. 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

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. [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?

43. 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 K 34, K -34, K 5, K -5 DNA binding affinity Rate constants (previously determined)

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. IEP K -34 K5K5 M S I Drivers of recombination inefficiency: 1. Low-affinity DNA-monomer binding

49. IEP K -34 K5K5 M S I Drivers of recombination inefficiency: 1. Low-affinity DNA-monomer binding 2. Synaptic stability

50. IEP K -34 K5K5 M S I Story of Flp: Low-affinity DNA-monomer binding requiring 10x more protein than Cre for DNA binding, yet also achieving 100% recombination.

51. IEP K -34 K5K5 M S I Story of Cre: High-affinity DNA-monomer binding requiring 10x less protein than Flp, yet achieving <75% recombination due to synaptic stability.

52. IEP K -34 K5K5 M S I Punchline. Likely an optimum that balance DNA binding affinity and synaptic stability.

53. Punchline. Parameters and mechanistic model establish a basis for incorporating recombination in dynamic model for counter architecture.