Stochastic and reversible assembly of a multiprotein DNA repair complex ensures accurate target site recognition and efficient repair

Computational modeling and quantitative analysis show that although accumulation of repair complexes can take hours, the individual components rapidly exchange between the nucleoplasm and DNA damage sites

Initial distribution of inflicted lesions (6-4 photoproducts) and final distribution of EdU incorporation match.

<p>(<b>A</b>) XP–C XPC-eGFP cells were locally irradiated and fixed immediately. The distribution of inflicted 6-4PP lesions was determined by immunostaining followed by scoring of the immunofluorescence signal in <i>n</i> = 250 cells from five experiments (<b>B</b>) XP–C XPC-eGFP cells were locally irradiated and cultivated for 4 hours in the presence of EdU before fixation. Distribution of EdU incorporation was plotted based on measurements derived from <i>n</i> = 198 scored local damages derived from three independent experiments.</p

EdU incorporation colocalizes with sites of local damage and reflects repair DNA synthesis quantitatively.

<p>(<b>A</b>) DNA in HeLa cells (upper panel) or XP–C XPC-eGFP cells (lower panel) was locally damaged by UV irradiation and subsequently incubated for 30 minutes in the presence of 10 µM EdU. Arrows indicate local damage areas as revealed by accumulation of endogenous XPC (upper panel) or stably expressed XPC-eGFP (lower panel); EdU incorporation is a measure for repair DNA synthesis. (<b>B</b>) Repair DNA synthesis on sites of local damage as determined by quantitative microscopy coincides with the removal of 6-4PPs measured previously in the same set-up by antibody staining <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003438#pcbi.1003438-Luijsterburg1" target="_blank">[8]</a>. Graphs display the means ± SD, <i>n</i> = 50–70 locally damaged cells per time point for damaged DNA and <i>n</i> = 150 locally damaged cells (derived from three independent experiments) per time point for repaired DNA. (<b>C</b>) Comparison of increase in EdU incorporation in locally damaged DNA regions with increase in PCNA accumulation as measured previously <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003438#pcbi.1003438-Luijsterburg1" target="_blank">[8]</a>. (<b>D</b>) Plotting the EdU data according to a linearization of <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003438#pcbi.1003438.e001" target="_blank">Eq. (1)</a> reveals a single-exponential time course.</p

Control over the rate of DNA repair predicted to be small and distributed over all repair proteins.

<p>(<b>A</b>) Response coefficients for the control of the repair rate by the concentrations of the repair factors. (<b>B</b>) Repair rate as a function of concentration changes in individual repair factors.</p

Variable expression levels of NER factors and inflicted DNA lesions quantitatively account for the distribution of repair rates.

<p>Comparison of modeled and measured correlation of nuclear XPC concentration and XPC accumulation in the locally damaged region (<b>A</b>) and between nuclear XPC concentration and DNA synthesis (<b>B</b>). Red lines represent linear regression with correlation coefficient <i>r</i> and <i>p</i>-value. (<b>C</b>) and (<b>D</b>) as in (A) and (B) but for XPA. (<b>E</b>) Simulated variability of the DNA repair kinetic under the influence of a single variable protein (dots) and of all variable components (triangles). For comparison the experimental <i>CV</i> is shown at five time points and error bars obtained with nonparametric bootstrap. (<b>F</b>) Time evolution of distribution of EdU incorporation: measured (red bars) versus simulated (blue lines). 95% confidence bounds of all correlation coeffiecients <i>r</i> were estimated by non-parametric bootstrap and are given in brackets.</p

Analytical model of the formation of a catalytic multi-protein complex on DNA.

<p>(<b>A</b>) Sequential (above) and random (below) assembly schemes for a complex of three components (A, B and C); x₀ denotes the empty assembly site (e.g., a DNA lesion), x_A the assembly site with component A bound etc. The complete complex (x_ABC) catalyses a reaction (e.g., lesion repair) with rate r. The repair process consists of several such assembly-reaction modules (cf. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003438#pcbi-1003438-g001" target="_blank">Fig. 1</a>). (<b>B</b>) The mean reaction time <i>τ</i> given by <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003438#pcbi.1003438.e002" target="_blank">Eq. (2)</a> depends on the number of assembling protein components <i>N</i> and on the assembly mechanism. Parameters: apparent on-rates <i>k</i> equal to off-rates (<i>k</i> = <i>l</i> = 1 min⁻¹), <i>ρ</i> = 10 s⁻¹ (<b>C</b>) In the case of reversible assembly (<i>k</i> = <i>l</i> = 1 min⁻¹, <i>N</i> = 9, random mechanism), the formation of the reaction product follows exponential kinetics (solid red line, fitted perfectly by a mono-exponential time course with time constant <i>τ</i>). Irreversible assembly (<i>l</i> = 0) follows a sigmoidal time course (dashed red line, <i>N</i> = 9, random mechanism, on-rate <i>k</i> = 0.037 min⁻¹ chosen to get the same time constant). The results for a sequential assembly mechanisms are qualitatively similar.</p

Natural expression variability of the repair factors XPC and XPA has small effect on DNA repair synthesis.

<p>(<b>A</b>) Frequency histograms of endogenous XPC (<i>n</i> = 1727) and XPA (n = 1462 cells) concentration in HeLa cells. (<b>B</b>) Scatter plot of XPC-eGFP fluorescence in XP–C XPC-eGFP cells against an antibody recognizing XPC (left, <i>n</i> = 332 cells) and eGFP-XPA fluorescence in XP–A eGFP-XPA cell line against an antibody recognizing XPA (right, n = 2142 cells) as determined by quantitative (immuno) fluorescence microscopy. (<b>C</b>) Scatter plot of endogenous XPC nuclear concentration versus endogenous XPC concentration in DNA damaged areas 30 minutes post-irradiation. (<b>D</b>) Endogenous XPA nuclear concentration versus endogenous XPA concentration in damaged areas 60 minutes post irradiation. (<b>E</b>) Scatter plot of endogenous XPC nuclear concentration versus repair DNA synthesis as measured by EdU incorporation on local damage 30 minutes post-irradiation (<i>n</i> = 303 in three independent experiments). (<b>F</b>) Scatter plot as in (<b>E</b>), but for XPA (n = 170 cells). Cells were analysed 60 minutes after inflicting UV damage, i.e. at maximum XPA accumulation. 95% confidence bounds of all correlation coeffiecients <i>r</i> were estimated by non-parametric bootstrap and are given in brackets.</p

Assembly of multiprotein complexes that control genome function

Live-cell imaging studies aided by mathematical modeling have provided unprecedented insight into assembly mechanisms of multiprotein complexes that control genome function. Such studies have unveiled emerging properties of chromatin-associated systems involved in DNA repair and transcription