PCNA ubiquitylation contributes to the interaction of Polδ with the clamp.

<p>(<b>A</b>) Assessing co-immunoprecipitation of Polδ and PCNA. Top: experimental scheme for co-immunoprecipitation experiments. Cells were either treated, or not, with hydroxyurea (HU) and TCA extracts prepared to monitor PCNA modification (whole cell) or soluble extracts prepared for immunoprecipitation with either anti-PCNA or anti-GFP (the catalytic subunit of Polδ is GFP tagged). Bottom: comparison of the Polδ - PCNA interaction in <i>rad18</i>Δ (Δ), wild-type (+) or <i>urg1-rad18</i> cells (++) cells that exhibiting distinct levels of PCNA ubiquitylation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006789#pgen.1006789.g002" target="_blank">Fig 2A</a>). (<b>B</b>) Cell-cycle profiles. Open histograms -HU, grey histograms +HU. (<b>C</b>) Quantification of modified forms of PCNA that was co-immunoprecipitated with Polδ (“IP with αGFP” in a) and in whole cell extracts. (<b>D</b>) Co-immunoprecipitation of Polδ in P_urg1-<i>rad18</i> cells is partially dependent on <i>ubc13</i>. (<b>E</b>) Quantification of DNA-associated Polδ by single molecule PALM imaging (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006789#pgen.1006789.g002" target="_blank">Fig 2F</a> for details). (<b>F</b>). Increased chromatin association of PCNA in the <i>elg1</i>Δ genetic background. Chromatin was fractionated from the indicated strains and probed for Pcn1 and a histone H3. (<b>G</b>) Co-immunoprecipitation of Polδ with PCNA in <i>elg1</i>⁺ (+) and <i>elg1</i>Δ (Δ) cells in the <i>rad18</i>⁺ and <i>rad18</i>Δ genetic backgrounds.</p

PCNA ubiquitylation mediates repair of ssDNA gaps during S-phase.

<p>(<b>A</b>) Schematic of the S1 nuclease assay [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006789#pgen.1006789.ref036" target="_blank">36</a>] used to monitor gaps during DNA replication. (<b>B</b>) Experimental scheme. <i>cdc25-22</i> cells were synchronised at G2 phase by temperature shift then released into the cell cycle and samples harvested at the indicated time points. (<b>C</b>) Analysis of S1-nuclease digested total DNA. Samples were subjected to agarose gel electrophoresis and visualised by ethidium bromide (EtBr) staining to reveal the total DNA (top) and subjected to BrdU antibody detection to reveal newly synthesised strands (bottom). (<b>D</b>) Quantification of S1-nuclese digested fragments. Fractions of fragments along the axis of fragment length were calculated from the intensity of BrdU signals in C. (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006789#pgen.1006789.s007" target="_blank">S7 Fig</a> for the calculation steps). (<b>E</b>) Schematic of the BrdU assay used to monitor gaps during DNA replication. (<b>F</b>) detection of BrdU incorporated into the indicated DNA samples prepared in agarose plugs. Experimental scheme as in B. (<b>G</b>) Quantification of BrdU incorporation. Plotted data are derived from three independent experiments.</p

PCNA ubiquitylation predominantly affects Polδ function.

<p><b>(A</b>) Spot tests showing the influence of PCNA ubiquitylation on cell growth/viability in genetic backgrounds compromised for Polε or Polδ function. <i>cdc20-m10</i> and <i>cdc6-23</i> are temperature sensitive mutations in the catalytic subunits of Polε and Polδ, respectively. (<b>B</b>) The effect of <i>pcn1-K164R</i> mutation and <i>rad18</i> deletion on the temperature sensitivity of <i>cdc6-23</i> (Polδ) are epistatic. (<b>C</b>) Model. Left: Schematic of Okazaki fragment synthesis. (i) Following priming by Polα-primase PCNA is loaded by RFC. (ii) Polδ associates with PCNA and synthesis begins. Rad18-Rad6 mono-ubiquitylates PCNA. (iii) Stochastic dissociation of Polδ from PCNA. Ubiquitylation prevents PCNA dissociation/unloading. (iv) Polδ re-associates with PCNA and synthesis resumes. (v) Okazaki fragment synthesis is completed. Right, a potential effect of loss of Rad18: (vi) Following priming by Polα-primaae PCNA is loaded by RFC. (vii) Polδ associates with PCNA and synthesis begins. (viii) Stochastic dissociation of Polδ from PCNA. (ix) PCNA is not ubiquitylated and thus can dissociate (possibly unloaded by Elg1). (x) Okazaki fragment synthesis is not completed and a gap remains.</p

PCNA ubiquitylation influences PCNA chromatin association.

<p><b>(A</b>) PCNA ubiquitylation levels in log phase cells when Rad18 is absent (Δ), expressed normally (+) or upregulated in P_urg1-<i>rad18</i> cells (++). Ubiquitylation was assessed following protein extraction in denaturing condition (TCA prep). (<b>B</b>) PCNA chromatin association. The cells shown in panel A expressing different levels of Rad18 and thus harbouring distinct level of PCNA ubiquitylation were analysed for PCNA chromatin association. Pgk1 (a soluble cytosolic protein) and histone H3 were used as controls. (<b>C</b>) Dependency of chromatin association on PCNA-K164 and Ubc13. An equivalent experiment as shown in panel B for <i>pcn1-K164R</i> and <i>ubc13</i> mutant backgrounds. (<b>D</b>) Quantification of the modified and unmodified PCNA from C. (<b>E</b>) Chromatin association of PCNA during S phase progression. Top left, experimental scheme: cells were synchronised at the G1/S boundary by temperature shift and PCNA chromatin association (bottom left panel) and cell cycle profiles (right) monitored at the indicated time points after release into S phase. The relative amounts of chromatin-associated PCNA were quantified (centre) (<b>F</b>) Quantification of DNA-associated PCNA by single molecule PALM imaging. Top left: schematic showing how motion blurring filters out mobile molecules. Bottom left: example of the visualisation of only the DNA-associated molecules. Middle: example of static localised molecule (top) and the typical signal from a diffusing molecule (bottom). Right: box plots of the numbers of visualised immobile PCNA molecules in the S phase nuclei of the indicated strains. Bi-nucleate (S phase) <i>mEos3-pcn1</i> cells (expressing mEos3-tagged PCNA) were imaged and mEos3-PCNA localisations quantified per nucleus.</p

The impact of PCNA ubiquitylation on genome replication.

<p>(<b>A</b>) Time-course of PCNA ubiquitylation during S-phase in fission yeast cells. Top-left, experimental scheme: cells were synchronised at the G1/S boundary, UV-irradiated, released from the arrest by incubation at 25°C and samples were analysed at the indicated time points for cell-cycle profile (top-right) and PCNA ubiquitylation status (bottom). As = asynchronous cells. (<b>B</b>) Experimental scheme to analyse replication dynamics by BrdU incorporation: fission yeast cells are synchronised in G2 and BrdU added to the media when cells were released. Samples are taken at 75 and 90 minutes for the experiments in panel D and at the indicated times for experiments in panels C and E. (<b>C</b>) Global BrdU incorporation during synchronous S-phase. Replication was analysed by detecting BrdU in purified total genomic DNA by dot-blot. (<b>D</b>) A representative region showing BrdU enrichment following immunoprecipitation and high throughput sequence analysis at two time points for <i>rad18</i>+ and <i>rad18</i>Δ cells progressing through S phase. (<b>E</b>) Replication profiles throughout S phase. Local replication extents were determined following BrdU immunoprecipitation and high throughput sequence analysis. The progression of local replication for each 300 bp chromosomal region was plotted for three conditions: when global genomic replication progression was either 25%, 50% or 75% complete. The blue line represents <i>rad18</i>^<i>+</i>, the red line <i>rad18</i>Δ. Filled shaded area between these lines highlight the regions where replication extents differ between the two strains; light blue—<i>rad18</i>⁺ > <i>rad18</i>Δ, light red—<i>rad18</i>⁺ < <i>rad18</i>Δ. (<b>F</b>) Ensemble analysis of replication timing at origins. The distribution of local replication progress when global genomic replication progress is either 25%, 50% or 75% complete. Left: early replicating regions (origins, indicated by open triangle in E). Right: late-replicating regions distal to the origins (indicated by inverted solid triangle in E). (<b>G</b>) Correlation between late replication and relative change in replication timing between <i>rad18</i>⁺ and <i>rad18</i>Δ. The latest replicating regions in <i>rad18Δ</i> cells are shown in pink (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006789#pgen.1006789.s004" target="_blank">S4 Fig</a>).</p

Malfunction of nuclease ERCC1-XPF results in diverse clinical manifestations and causes Cockayne syndrome, xeroderma pigmentosum, and Fanconi anemia

Cockayne syndrome (CS) is a genetic disorder characterized by developmental abnormalities and photodermatosis resulting from the lack of transcription-coupled nucleotide excision repair, which is responsible for the removal of photodamage from actively transcribed genes. To date, all identified causative mutations for CS have been in the two known CS-associated genes, ERCC8 (CSA) and ERCC6 (CSB). For the rare combined xeroderma pigmentosum (XP) and CS phenotype, all identified mutations are in three of the XP-associated genes, ERCC3 (XPB), ERCC2 (XPD), and ERCC5 (XPG). In a previous report, we identified several CS cases who did not have mutations in any of these genes. In this paper, we describe three CS individuals deficient in ERCC1 or ERCC4 (XPF). Remarkably, one of these individuals with XP complementation group F (XP-F) had clinical features of three different DNA-repair disorders - CS, XP, and Fanconi anemia (FA). Our results, together with those from Bogliolo et al., who describe XPF alterations resulting in FA alone, indicate a multifunctional role for XPF. © 2013 The American Society of Human Genetics.</p

Clinical features of ATR/ATRIP–deficient patients.

<p>NR; not recorded. NA; not assessed.</p

MGS and Seckel syndrome patient phenotypes.

*<p>standard deviations from the age-related normal population mean, NA = not assessed.</p><p>MGS data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945-Bicknell1" target="_blank">[13]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945-Bicknell2" target="_blank">[14]</a><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945-Guernsey1" target="_blank">[33]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945-deMunnik1" target="_blank">[34]</a>.</p

ALC1/CHD1L, a chromatin-remodeling enzyme, is required for efficient base excision repair

<div><p>ALC1/CHD1L is a member of the SNF2 superfamily of ATPases carrying a macrodomain that binds poly(ADP-ribose). Poly(ADP-ribose) polymerase (PARP) 1 and 2 synthesize poly(ADP-ribose) at DNA-strand cleavage sites, promoting base excision repair (BER). Although depletion of ALC1 causes increased sensitivity to various DNA-damaging agents (H₂O₂, UV, and phleomycin), the role played by ALC1 in BER has not yet been established. To explore this role, as well as the role of ALC1’s ATPase activity in BER, we disrupted the <i>ALC1</i> gene and inserted the ATPase-dead (E165Q) mutation into the <i>ALC1</i> gene in chicken DT40 cells, which do not express PARP2. The resulting <i>ALC1</i>^<i>-/-</i> and <i>ALC1</i>^{<i>-/E165Q</i>} cells displayed an indistinguishable hypersensitivity to methylmethane sulfonate (MMS), an alkylating agent, and to H₂O₂, indicating that ATPase plays an essential role in the DNA-damage response. <i>PARP1</i>^<i>-/-</i> and <i>ALC1</i>^<i>-/-</i><i>/PARP1</i>^<i>-/-</i> cells exhibited a very similar sensitivity to MMS, suggesting that ALC1 and PARP1 collaborate in BER. Following pulse-exposure to H₂O₂, <i>PARP1</i>^<i>-/-</i> and <i>ALC1</i>^<i>-/-</i><i>/PARP1</i>^<i>-/-</i> cells showed similarly delayed kinetics in the repair of single-strand breaks, which arise as BER intermediates. To ascertain ALC1’s role in BER in mammalian cells, we disrupted the <i>ALC1</i> gene in human TK6 cells. Following exposure to MMS and to H₂O₂, the <i>ALC1</i>^<i>-/-</i> TK6 cell line showed a delay in single-strand-break repair. We therefore conclude that ALC1 plays a role in BER. Following exposure to H₂O_2, <i>ALC1</i>^<i>-/-</i> cells showed compromised chromatin relaxation. We thus propose that ALC1 is a unique BER factor that functions in a chromatin context, most likely as a chromatin-remodeling enzyme.</p></div

CV1720 cells show impaired ATR–dependent DNA damage responses.

<p>A) WT, DK0064 (ATR–SS), CV1720 (patient), CV1780 (patient's mother) and CV1783 (patient's father) cells were exposed to 5 Jm⁻² UV and the mitotic index (MI) assessed 2 h post exposure. A greater than two fold decrease in mitotic index is observed in WT and both paternal cell lines but not in DK0064 (ATR–SS) or CV1720 (patient) cells. B) Cells were exposed to 5 mM HU for 2 h and the percentage of p-H2AX (γ-H2AX) positive cells assessed by immunofluorescence. Note that HU causes pan nuclear p-H2AX formation rather than defined foci as observed after exposure to ionising radiation. Thus, the percentage of γ-H2AX positive cells was scored. C) Cells were exposed to UV (5 Jm⁻²) and subjected to Western Blotting (WB) using p-Chk1 (p-Ser317) antibodies at 2 h. Chk1 expression was shown to be similar in WT and patient cells (lower panel). D) Cells were exposed to 3 mM HU for 2 h and whole cell extracts analysed by WB using FANCD2 antibodies. The ubiquitylation of FANCD2, detectable by a product with reduced mobility, is diminished in DK0064 (ATR–SS) and CV1720 cells compared to WT cells. E) Cells were exposed to 5 mM HU and examined for the percentage of cells showing >5 53BP1 foci at 2 h post exposure. 53BP1 foci formation is reduced in DK0064 (ATR–SS) and CV1720 cells compared to WT cells. F–I) The indicated cells were processed by WB using ATRIP or ATR antibodies. MCM2 was used as a loading control. F shows the analysis of a range of protein levels for accurate comparison. CV1720 (patient) cells show markedly reduced ATR and ATRIP protein levels. G shows that both parental lines have approximately half the level of ATR and ATRIP compared to two WT cell lines. DK0064 (ATR–SS) and CV1720 cells, in contrast, have more dramatically reduced ATR and ATRIP protein levels. 50 ug protein was loaded. WT in all panels was GM2188. Patient, mother and father were as shown in panel A. H and I show the quantification of ATRIP and ATR protein levels from at least three independent WB experiments.</p