15 research outputs found

    Complete reconstitution of XPA<sup>−/−</sup> cells requires XPA with intact APIM.

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    <p>(A) Cell proliferation after UV-B treatment measured by MTT assay. The data is normalized against untreated day 1. One representative out of three experiments is presented. Data presented is the average of 6 wells ± SD. (B) Normalized XPA intensity measured by in-cell western (LI-COR Bioscience) (mean ± SD, n = 6). The XPA intensity is normalized against the DNA content using Draq5. (C) <i>Left panel:</i> Histograms of 6-4 PP positive cells, untreated, and 0, 2 and 4 h after UV-B. The cells with fluorescent intensity above the dashed line are defined as 6-4 PP positive. The numbers in the bottom row indicate % 6-4 PP positive cells 4 h after UVR. <i>Right panel:</i> Graphic presentation of data in left panel showing reduction of 6-4 PP positive cells as a function of time. (D) <i>Left panel:</i> Histograms illustrating cell cycle distribution of CPD positive and negative cells, untreated, 0 and 24 h after UV-B. Lower UVR-dose was applied for the XPA<sup>−/−</sup> cells to avoid excessive apoptosis. The dashed lines separate the cell cycle phases. % CPD positive cells are given in bottom row. <i>Right panel</i>: Bars illustrating the relative cell-phase distribution of the CPD positive cells.</p

    The APIM sequence in XPA is sufficient and necessary for interaction with PCNA.

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    <p>(A) Sequence alignment of the APIM sequence in XPA (aa 161–170 in human XPA) from different species compared with the APIM sequence in hABH2. The colors are given by Clustal X. (B) Dot blot with the human XPA APIM-peptide. The hABH2 APIM-peptide and its mutant are included as positive and negative controls, respectively (also used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049199#pone.0049199-Gilljam1" target="_blank">[27]</a>). Grey lines: dots from the same blot. (C) Images of YFP-tagged XPA<sub>161−167</sub> co-expressed with CFP-tagged PCNA in live cycling HeLa cells. Yellow dots in the merged picture illustrate colocalization. Bar: 5 µM. (D and E) N<sub>FRET</sub> measurements in HeLa cells. Detector gain: 800 (YFP), 700 (CFP), 700 (FRET) (D) and 700 (YFP), 800 (CFP), 700 (FRET) (E). CFP/YFP (vectors only) and CFP-PCNA/YFP-PCNA were used as negative and positive controls, respectively (mean ± SEM, n = 24–53 in D and n = 10–34 in E). (F) Overexpressed tagged proteins in live cycling XPA<sup>−/−</sup> cells. Yellow dots in the merged picture illustrate colocalization. Bar: 5 µM. (G). N<sub>FRET</sub> measurements in XPA<sup>−/−</sup> cells. Detector gain: 800 (YFP), 700 (CFP), 700 (FRET) (mean ± SEM, n = 25–66). The P-values (D, E and G) are derived by unpaired t-test.</p

    Model describing the role of direct XPA-PCNA interaction for efficient NER after UVR.

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    <p>To clarify the essence of our hypothesis, only the XPA dimer, XAB2, and RPA of the NER proteins are specified, and the NER complex (yellow) represents the other NER proteins in the model. The grey proteins mark proteins containing the PIP-box, the green mark proteins containing APIM, the blue donut marks PCNA and the red hooks mark 6-4 PPs and CPDs. (A) Optimal NER. (B) Reduced NER due to mutated APIM sequence in XPA.</p

    Nucleotide Excision Repair Is Associated with the Replisome and Its Efficiency Depends on a Direct Interaction between XPA and PCNA

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    <div><p>Proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication, DNA repair, cell cycle regulation, chromatin remodeling, and epigenetics. Many proteins interact with PCNA through the PCNA interacting peptide (PIP)-box or the newly identified AlkB homolog 2 PCNA interacting motif (APIM). The xeroderma pigmentosum group A (XPA) protein, with a central but somewhat elusive role in nucleotide excision repair (NER), contains the APIM sequence suggesting an interaction with PCNA. With an in vivo based approach, using modern techniques in live human cells, we show that APIM in XPA is a functional PCNA interacting motif and that efficient NER of UV lesions is dependent on an intact APIM sequence in XPA. We show that XPA<sup>−/−</sup> cells complemented with XPA containing a mutated APIM sequence have increased UV sensitivity, reduced repair of cyclobutane pyrimidine dimers and (6–4) photoproducts, and are consequently more arrested in S phase as compared to XPA<sup>−/−</sup> cells complemented with wild type XPA. Notably, XPA colocalizes with PCNA in replication foci and is loaded on newly synthesized DNA in undamaged cells. In addition, the TFIIH subunit XPD, as well as XPF are loaded on DNA together with XPA, and XPC and XPG colocalize with PCNA in replication foci. Altogether, our results suggest a presence of the NER complex in the vicinity of the replisome and a novel role of NER in post-replicative repair.</p> </div

    XPA colocalizes and directly interacts with PCNA in replication foci

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    <p>. (A) Overexpressed tagged proteins in live cycling HeLa cells. (B) Immunostained HeLa cells. The intensity of α-XPA and α-PCNA along the line in the merged picture is illustrated in the graph. The inserts show an enlargement of the area close to foci 3 and 4. (A and B) Bar: 5 µm. (C) iPOND from cells labeled with EdU (pulse) before fixation. One sample was additionally followed by a chase in thymidine-containing medium (pulse-chase). The WB shows proteins captured due to EdU proximity. The upper and lower panels are from individual iPOND experiments. All bands within one panel (black frame) are from the same WB, lanes and rows are separated by grey lines (also in D and E). (D) Co-IP of endogenous XPA from HeLa cells stably expressing YFP-PCNA using α-YFP beads. SF: soluble fraction, CF: chromatin-enriched fraction, Y: YFP (negative control), Y-P: YFP-PCNA. (E) Co-IP of endogenous XPA from untransfected HeLa cells using α-PCNA beads (pulling down endogenous PCNA). IP with α-YFP was used as control for unspecific binding to the beads. (F) Normalized FRET (N<sub>FRET</sub>) measurements in HeLa cells. CFP/YFP (vectors only) and CFP-PCNA/YFP-PCNA were used as negative and positive controls, respectively. Detector gain: 800 (YFP), 700 (CFP), 700 (FRET). The P-value is derived by unpaired t-test. Data presented is from three independent experiments (mean ± SEM, n = 55–75).</p

    After UVR, cells complemented with APIM-mutated XPA accumulate γH2AX foci at the site of replication.

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    <p>(A) Normalized γH2AX intensity measured by in-cell western (LI-COR Bioscience) (mean ± SD, n = 4) 24 h after exposure to UV-B. The γH2AX intensity is normalized against the DNA content using Draq5 and the intensity of untreated cells. (B) Images of immunostained cells. The cells were exposed to UV-B 24 h prior to fixation. Lower UVR-dose was applied for the XPA<sup>−/−</sup> cells to avoid excessive apoptosis. Bar: 5 µm. (C) Fractions of replication foci (PCNA) colocalizing with γH2AX. Each dot represents one cell, on average 35 foci were counted in each cell (mean ± SEM, n = 5 and 15). The P-value is derived by unpaired t-test. Only cells resembling S phase cells and expressing comparable levels of the YFP constructs were included.</p

    Melphalan-resistant cells have a more rapid kinetics of γH2AX foci formation than sensitive cells.

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    <p>(A) The number of γ-H2AX foci per cell nucleus (N>13) was manually quantified for each time point and the experiment was performed in triplicate. Corresponding standard deviation bars are indicated. P<0.01 at all time points as calculated using 2-sample t-test with equal variance. (B) Representative confocal images of the cells at different time points subsequent to Melphalan treatment. Foci formation was monitored using γH2AX polyclonal antibody (red) and due to the significant fraction of NFkB protein present in the cytoplasm of these cells, NFkB p65 mouse monoclonal antibody was used as control for cytoplasmic staining (green).</p

    An Inverse Switch in DNA Base Excision and Strand Break Repair Contributes to Melphalan Resistance in Multiple Myeloma Cells

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    <div><p>Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs). Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel candidate biomarkers for Melphalan sensitivity that will be included in targeted quantitation studies in larger patient cohorts to validate their value in prognosis as well as targets for replacement- or adjuvant therapies.</p> </div

    Steady state expression levels of proteins involved in DNA repair and DNA damage signaling response in 8226-LR5 cells relative to parental cells.

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    <p>Protein levels were assessed by quantitative western blot analysis using specific antibodies against target proteins. Each bar represent the mean expression ratio of the target protein in the LR5 (resistant) cells relative to the reference value of 1 (dotted line) in the sensitive cells subsequent to normalization against either β-actin or tubulin (to avoid overlapping signals). Quantitative analysis enclosed an average of 3 to 5 biological replicates with standard deviations as indicated. The P values were calculated by one sample two tailed t test against a hypothetical expression ratio set to 1 (no change in expression). >90%, >95% and >98% confidence levels indicated by *, **, and ***, respectively. # Only non-ubiquitinylated FANCD2 was detected in the analyses.</p

    Effect of PI3-kinase-like kinase (PIKK)- and CHK1/2 inhibitors on the proliferation of 8226-LR5 cells.

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    <p>The MTT assay was used to monitor the survival after 72h incubation with the various inhibitors ±2.5 Melphalan. CHK1/2 inhibitor AZD7762 (0.1 µM), DNA-PK inhibitor NU7441 (0.2 µM), ATM inhibitor KU55933 (3 µM), ATR inhibitor VE821 (1 µM). Whereas a moderate inhibition on proliferation was observed with either inhibitor alone, strong inhibition was observed in combination with melphalan for the CHK1/2, DNA-PK and ATR inhibitors. Each bar represents the mean of at least 5 independent experiments with standard deviations as indicated. P-values were calculated using 2-sample t-test with equal variance. >95%, >99% and >99.9% confidence levels indicated by *, **, and ***, respectively.</p
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