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

<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^−/− 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^−/− 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

Complete reconstitution of XPA^−/− cells requires XPA with intact APIM.

<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^−/− 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.

<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_161−167 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_FRET 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^−/− cells. Yellow dots in the merged picture illustrate colocalization. Bar: 5 µM. (G). N_FRET measurements in XPA^−/− 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.

<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

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

<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^−/− 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

XPA colocalizes and directly interacts with PCNA in replication foci

<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_FRET) 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

Schematic representation of yeast two-hybrid high confidence hits involved in transcription.

<p>Individual prey fragment clones and the resulting selected interaction domains (SIDs) reported to bind ALKBH4 are indicated above each protein; black lines, placenta library; orange lines, fetal brain library; green lines, placenta library screened with ALKBH4^H169A/D171A; red dashed lines, SIDs. Grey boxes indicate protein domains. Proteins and domains are drawn to scale according to the InterPro (version 4.8) and PROSITE (release 20.68) databases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049045#pone.0049045-Hunter1" target="_blank">[73]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049045#pone.0049045-Sigrist1" target="_blank">[74]</a>.//indicates regions omitted for simplicity. ZnF, C₂H₂ zinc finger; HD, homeodomain; YEATS, Yaf9 ENL AF9 Taf14 Sas5; TAZ, transcription adaptor putative zinc finger; BRD, bromodomain; PHD plant homeodomain; HAT histone acetyl transferase; DBD, DNA binding domain. Bar, 100 aa.</p

The genes most differentially expressed in either direction identified in ALKBH7 over-expressing cells compared to parental non over-expressing cells.

<p>The genes most differentially expressed in either direction identified in ALKBH7 over-expressing cells compared to parental non over-expressing cells.</p

Ectopic ALKBH4 expression does not change H3K79 methylation levels <i>in vivo</i>.

<p>As analyzed by Western blotting, all three methylation states (mono- di- and tri-methylation) of the H3K79 residue remained similar in histones purified from stable HEK293 transfectants after doxycycline-dependent over-expression of either ALKBH4 or an enzymatically inactive mutant (ALKBH4^H169A/D171A), compared to the equivalent, non-induced cells. Signal intensities of bands corresponding to methylated histones were quantified using ImageJ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049045#pone.0049045-Schneider1" target="_blank">[69]</a>, and normalized to the total histone H3 load, but no effect of ALKBH4 overexpression was detected (not shown).</p

High confidence^a ALKBH4 interacting proteins identified by the yeast two-hybrid system.

a<p>PBS (predicted biological score) of A or B. PBS (calculated according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049045#pone.0049045-Formstecher1" target="_blank">[75]</a>) represents the probability of an interaction to be non-specific, and refers to an e-value with defined thresholds to rank the results in the high-to-low confidence categories A–D.</p>b<p>SID (selected interaction domain) refers to the amino acid region shared by all prey fragments matching the same reference protein,</p>c<p>(+) Low confidence hit (PBS D),</p>d<p>EIF3CL. Grey shade, transcription related interactants. YEATS, Yaf9 ENL AF9 TAF14 Sas5; DBD, DNA binding domain; PHD, plant homeodomain; PET, Prickle Espinas Testin; LIM, Lin-11 Isl-1 Mec-3; PCI, Proteasome, COP9, Initiation factor-3; PTP, protein tyrosine phosphatase.</p