INTRODUCTION

1. Int IHF Xis + Fis Int IHF Investigating Site-Specific Recombination of Bacteriophage Lambda Using Biochemical and Mathematical Approaches Steven Esquivel, Yi-An Lai, Ryan Blanchet, Kenny Sokolowski, Peter Salamon‡, AncaSegall‡ - San Diego State University, San Diego, California ‡ Co-Senior Author RESULTS Modeling The Four Dimension Differential Equation System INTRODUCTION RESULTS Bacteriophage lambda lytic cycle OBJECTIVES CONCLUSIONS Determine the rate constants of the steps in phage lambda site-specific recombination. • TS junction isomer is biased toward reverse resolution. • BS junction isomer is biased toward forward resolution. • WT junction isomer resolves to attP/attB (E) greater than attL/attR (A), which is similar to BS junction isomer. Fig. 5.Simulation of the four dimension differential equation system solved using the Matlab ode23 command. We are utilizing a four dimension differential equation system by using a four by one column vector dydt(1,1)dydt(4,1), where (1,1) represents the first row and first column of a matrix we call dydt. The matrix dydt is thus a column vector (4 x 1) – (rows x columns) that consists of the 4 differential equations dA/dt  dE/dt. Fig. 1.Schematic view of excision recombination. Two DNA substrates (A) recombine resulting in a HJ intermediate (B & C) that may resolve to form products depicted as E. WT1 TS2 BS1 FUTUREDIRECTIONS NC 15 30 60 90 105 120 135 150 165 180 200 300 NC 15 30 60 90 105 120 135 150 165 180 200 300 NC 15 30 60 90 105 120 135 150 165 180 200 300 • Compare experimental data with another mathematical approach using Matlab. From this, we should be able to run computational simulations of this event using four differential equations A(t), B(t), C(t) and E(t). From this, we can incorporate actual values at specific time points and solve the differential equations to find the relative equilibrium. • Optimize conditions for the separation of the different Holliday junction isomers complexed with proteins, in order to simplify and to extend our analysis. HJ • Bacteriophage lambda integrates and excises its genomic DNA from bacteria cells via site-specific recombination. • Integrase (Int), a tyrosine recombinase, catalyses phage λ site-specific recombination (both integration & excision). • Accessory proteins: Integration host factor (IHF) bends DNA and excisionase (Xis) stimulates the excision reaction while inhibiting integration. attP NC attL attP attL Catalytic event in site-specific recombination attB Fig. 2.HJ resolution assay. Resolution reaction starting with HJ (B & C) as substrates. We have isolated the HJ intermediates, re-load them with proteins, and monitor the second round of catalytic events or the reverse of the first round of catalytic events. ACKNOWLEDGEMENTS This project has been funded by NSF grant 0827278 Interdisciplinary Training in Biology and Mathematics to Dr. A. M. Segall and Dr. P. Salamon, and by NIH grant R01-GM52847 to Dr. A. M. Segall. Cre R173K/loxP-HJ • X-ray crystal structure of Cre complexes with loxP site holliday junction (HJ) with symmetry. • Resolution: 2 Å • Cre protein is a prototypical member in tyrosine recombinase family. Fig. 3.Quantification of HJ resolution assay (Fig 2). The quantification data was fitted to models that solve for the forward (dE/dt) and reverse rate (dA/dt). Keq = k2f / k2r = C / B, 1.dA(t)/dt = (T/(1+ Keq)) k1r – A k1f 2.dT(t)/dt = A k1f – (T/(1+ Keq)) k1r – T Keq/(1+ Keq)k3f 3.dE(t)/dt = T Keq/(1+ Keq) k3f C E B A K. Ghosh and G. Van Duyne, Univ. of Pennsylvania Table 1.Ratios of E vs. A for the three different species of Holliday Junction. The table includes the proportions (dE/dA) of different HJ and the comparison of their relative equilibrium constants with one another. Fig. 4.Three dimension differential equation system used to represent the mechanism steps of the recombination reaction.