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

Example of polymer conformations of the adapted DL model at different looping regimes.

<p>(<b>A</b>) Conformation of the adapted-DL polymer with high short-range (p_short = 0.12) and low long-range (p_long = 0.04) looping probabilities and (<b>B</b>) the same polymer with low short-range (p_short = 0.04) and high long-range (p_long = 0.12) looping probabilities. The colour code labels the monomers of the polymer according to the visible spectrum along the length of the polymer. The inset in panel A displays the same situation as in (A) after abolishing all long-range looping (p_long = 0), showing that a uniform thick fibre is formed. More adapted DL polymer conformations with the same looping probabilities as shown in (A) and (B) are shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003877#pcbi.1003877.s005" target="_blank">Figure S5</a>. (<b>C</b>) Conformation of the domain-adapted DL polymer with high short-range (p_short = 0.16) and low long-range (p_long = 0.02) looping probabilities and (<b>D</b>) the same polymer with low short-range (p_short = 0.02) and high long-range (p_long = 0.16) looping probabilities. Here, topological domains are labelled red and non-looping linker regions blue.</p

Knock-down of CTCF and Rad21.

<p>The nuclear fraction of control cells (04-147) and cells depleted of CTCF, Rad21 or both simultaneously were analysed by SDS-PAGE followed by immunoblotting. β-Actin was used as a control when calculating changes in CTCF and Rad21 protein levels after siRNA-mediated knock down. Average protein amounts of at least three independent experiments are shown. Error bars represent standard deviation.</p

Relationship between looping probability and number of established loops.

<p>When keeping the long-range looping probability (p_long) constant while varying short-range looping probability (p_short), the number of long-range loops (n_long) increases when reducing p_short. This is because lowering p_short reduces the loop repulsion on the short scale and hence increases intermingling on the long scale, which in turn increases the frequency of long-range loop formation. Here, p_long = 0.06 and p_short is varied from 0.02 to 0.16. The blue area marks the short-range looping regime (<50 monomers) while the rest describes the long-range looping.</p

<i>In situ</i> chromatin structure.

<p>(<b>A, B</b>) The genomic context of analysed regions of the q-arms of human chromosome 1 (<b>A</b>) and chromosome 11 (<b>B</b>) is illustrated by the human transcriptome map. Vertical lines represent protein coding genes, the length depicting the average transcription level in a number of human tissues and cell lines <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003877#pcbi.1003877-Caron1" target="_blank">[42]</a>. The location of BAC probes used for FISH labelling are indicated above the maps. Arrows above the maps designate the regions where spatial distances between pairs of BAC probes were measured; the start of the arrow indicating the position of the reference BAC probe. On chromosome 1q three regions were analysed. Two 3 Mb regions were selected in a gene-rich and transcriptionally active region (green box) and in a region characterized by low gene density and low transcription (red box). (<b>C–F</b>) Graphs show the mean square physical distances <<i>R</i>²> between the FISH probes as a function of the genomic distance <i>g</i> for a 3 Mb gene-rich (<b>C</b>) and a gene-poor (<b>D</b>) region on chromosome 1 q-arm. Longer regions of chromosome 1 (30 Mb) (<b>E</b>) and of chromosome 11 (76 Mb) (<b>F</b>) were also analysed. Each data point represents an average of 30–150 distance measurements in at least two independent experiments. Error bars represent standard errors. Graphs are shown for control cells (black dots) and cells after knockdown of CTCF (green diamonds), of cohesin subunit Rad21(blue triangles) and of CTCF and Rad21 simultaneously (red squares). Results show that chromatin becomes more compact after knockdown of CTCF and Rad21 (cohesin). (<b>G, H</b>) Radial positions of BAC probes in nuclei for chromosome 1q (G) and chromosome 11q (H). Radial nuclear position of a BAC probe was calculated the distance between the centre of gravity of a FISH labelled genomic site and the centre of gravity of the nucleus and dividing that distance with the length of a straight line drawn from the nuclear centre to the nuclear envelope through the centre of the gravity of the labelled site. Error bars represent standard errors. Note that some chromatin regions were poorly labelled in CTCF and/or cohesin knock down conditions and therefore omitted from the graph.</p

Heat plots showing how polymer compaction varies in the p_short - p_long parameter space.

<p>Polymer compaction is represented as MSD plateau level as shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003877#pcbi-1003877-g004" target="_blank">Figure 4</a>. (<b>A</b>) adapted DL polymer, (<b>B</b>) domain-adapted DL polymer. The arrows in (A) show that decreasing the short-range looping probability (red arrow) results in compaction, while reducing the long-range looping probability (green arrow) leads to de-compaction. In the heat plot red marks compaction, green decompaction.</p

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

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