Volume 19, Issue 5, Pages 679-690 (September 2005) Mathematical Modeling of Nucleotide Excision Repair Reveals Efficiency of Sequential Assembly Strategies  Antonio Politi, Martijn J. Moné, Adriaan B. Houtsmuller, Deborah Hoogstraten, Wim Vermeulen, Reinhart Heinrich, Roel van Driel  Molecular Cell  Volume 19, Issue 5, Pages 679-690 (September 2005) DOI: 10.1016/j.molcel.2005.06.036 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Nucleotide Excision Repair Model and Recruitment Kinetics of XPC-HR23B (A) Scheme of the basic model showing the sequential binding of five different protein factors to a damaged DNA site and their simultaneous release after repair. (B) Recruitment kinetics of XPC-HR23B. Prior to UV irradiation, cell nuclei exhibited a uniform distribution of XPC-GFP (t = 0 s). After UV irradiation, images were captured at the indicated time points. XPC-GFP gradually accumulated in the nuclear area that was UV damaged (the pore in the mask is not shown but its position is indicated by the arrowheads). The bar represents 10 μm. (C–E) Accumulation at local UV damaged sites of XPC-HR23B, TFIIH, and ERCC1-XPF, respectively (error bars represent standard deviation [SD] between three, ten, and nine different experiments, respectively. Circles represent their average). The red and black lines are model predictions with Parameter Set 1 and 2 from Table 1, respectively (the basic model and taking into account free factor diffusion, Supplemental Data section Basic Model Including Diffusion). In the simulations, DNA repair is initiated by induction of 38 μM damages in an area that corresponds to about one-seventh of the nuclear volume (∼1 × 106 damaged DNA sites). (F and G) Simulated immobilized fraction of all repair factors with Parameter Set 1 and Set 2, respectively (Table 1). In (G), the recruitment kinetics of ERCC1-XPF (F5) are significantly shorter than of XPC-HR23B (F1) and TFIIH (F2). Immobilization of the limiting factor F3 is more rapid and more complete than that of other factors. (H) Measured binding and release of ERCC1-XPF after local UV irradiation (circles). The solid line is a model simulation with Parameter Set 2 (Parameter Set 1 gives similar results). Shown are also the total number of damaged DNA sites as a function of time (dashed line) and the calculated release kinetics of ERCC1-XPF in case of a higher damage dose (dotted line, initial 48 μM damaged DNA sites, which correspond to ∼1.2 × 106 lesions). The arrow indicates the value of the repair time τR obtained from Equation (9). In all panels, the data for TFIIH and ERCC1-XPF were rescaled so that their maximum values correspond to the immobilized fractions estimated from FRAP experiments, i.e., 50% and 35%, respectively (Hoogstraten et al., 2002; Houtsmuller et al., 1999). For XPC-HR23B, a high degree of immobilization is assumed (90%), ensuring saturating conditions (see text). Molecular Cell 2005 19, 679-690DOI: (10.1016/j.molcel.2005.06.036) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Repair Efficiency in the Basic Model (A) Fold changes in repair time τR due to changes in each of the individual rate constants. The repair time is calculated from Equation (9) for an initial damage given to the whole nucleus (concentration of 4 μM, i.e., 0.72 × 106 lesions). The fold changes are relative to the reference parameter values kref and reference repair time τRref (=20.56 min) obtained from Parameter Set 1, Table 1. τR reflects the repair efficiency in the sense that an increase in efficiency corresponds to a decrease in repair time. (B) Fold changes in τR due to changes of each of the total NER factor concentrations. (C) Changes in maximal repair rate Vmax due to variations in the total concentrations of all NER factors simultaneously. We increased or decreased the total concentrations by an equal factor. For fold changes equal to 1, the concentrations are as given in Table 1, Set 1. For a slow repair step (kR = 0.001 s−1, upper dashed line, or 0.007 s−1, solid line), Vmax depends nearly linearly on the factor concentrations (first order reaction). For a fast repair step (kR = 0.7 s−1, lower dashed line), in which repair is limited by PC assembly, the dependency on factor concentrations is of the second order. The values for Vmax have been normalized. Molecular Cell 2005 19, 679-690DOI: (10.1016/j.molcel.2005.06.036) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Random Binding of Repair Factors and Early Release of the First Factor (A) Five possible assembly mechanisms for the formation of the PC. Mechanism I–IV describe the random reversible binding of, in each case, two factors at a different step of the repair process. As in the basic model, after removal of the damage all factors are released. For clarity we do not show the intermediary complexes. We assume that all factors bind equally well (same association and dissociation rate constants) to their respective binding sites and have equal total concentrations. This avoids bias, due to differences in the affinities or concentrations, when comparing the different mechanisms. (B) Repair rate versus total damage concentration. The colors and roman numbers refer to the assembly mechanisms shown in (A). Inset is a magnification. For mechanism I, repair is inhibited at high damage (red line). Parameters are as follows: each total factor concentration is 0.2 μM, binding rate constant is 1 μM−1s−1, dissociation rate constant is 0.005 s−1, and the repair rate constant is 0.007 s−1. Molecular Cell 2005 19, 679-690DOI: (10.1016/j.molcel.2005.06.036) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Early Release of the First Factor (A) Dissociation of the first factor F1 at different steps of the assembly process. At step six all factors are released simultaneously (basic model). (B) Maximal repair rate as a function of the step where the first factor is released (shown in [A], dark encircled numbers). When the first factor is released before the other factors (step 5 to 1), the maximal repair rate increases. Parameter Set 1 of Table 1 is used, except T1 = 0.3 μM. (C) Residence times are calculated for saturating conditions (Supplemental Data section Repair Time and Residence Time) at the different steps where the first factor is released. Only when the first factor is released before the other factors does its residence time fit with the experimental data (Supplemental Experimental Procedures). Molecular Cell 2005 19, 679-690DOI: (10.1016/j.molcel.2005.06.036) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Preassembly of Repair Factors (A) Repair factors are allowed to reversibly associate in solution to form intermediary complexes and the holocomplex in a damage-independent fashion (shown for the case of three protein factors). The holocomplex (H) contains all factors and is able to repair DNA damage through a single binding step and a single repair step. (B) Repair time of the holocomplex ( τRh, solid lines) and of the sequential assembly mechanism ( τRs, dashed line) as a function of the number of factors involved (n) for nonsaturating damage. For the holocomplex, both weak and strong factor interactions were examined (Kh = 200 nM and Kh = 10 nM, respectively). Parameters are as follows: factors have equal concentrations T = 0.2 μM. The binding rate constants and repair rate constant of the holocomplex and single factors are k = 0.3 μM−1s−1 and kR = 0.007 s−1. Molecular Cell 2005 19, 679-690DOI: (10.1016/j.molcel.2005.06.036) Copyright © 2005 Elsevier Inc. Terms and Conditions