Cell cycle-specific UNG2 phosphorylations regulate protein turnover, activity and association with RPA

Human UNG2 is a multifunctional glycosylase that removes uracil near replication forks and in non-replicating DNA, and is important for affinity maturation of antibodies in B cells. How these diverse functions are regulated remains obscure. Here, we report three new phosphoforms of the non-catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23 likely optimises the protein to excise uracil along with rapidly moving replication forks. Our findings may aid further studies of how UNG2 initiates mutagenic rather than repair processing of activation-induced deaminase-generated uracil at Ig loci in B cells

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

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

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

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

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

Repair of U/G and U/A in DNA by UNG2-associated repair complexes takes place predominantly by short-patch repair both in proliferating and growth-arrested cells

Nuclear uracil-DNA glycosylase UNG2 has an established role in repair of U/A pairs resulting from misincorporation of dUMP during replication. In antigen-stimulated B-lymphocytes UNG2 removes uracil from U/G mispairs as part of somatic hypermutation and class switch recombination processes. Using antibodies specific for the N-terminal non-catalytic domain of UNG2, we isolated UNG2-associated repair complexes (UNG2-ARC) that carry out short-patch and long-patch base excision repair (BER). These complexes contain proteins required for both types of BER, including UNG2, APE1, POLβ, POLδ, XRCC1, PCNA and DNA ligase, the latter detected as activity. Short-patch repair was the predominant mechanism both in extracts and UNG2-ARC from proliferating and less BER-proficient growth-arrested cells. Repair of U/G mispairs and U/A pairs was completely inhibited by neutralizing UNG-antibodies, but whereas added recombinant SMUG1 could partially restore repair of U/G mispairs, it was unable to restore repair of U/A pairs in UNG2-ARC. Neutralizing antibodies to APE1 and POLβ, and depletion of XRCC1 strongly reduced short-patch BER, and a fraction of long-patch repair was POLβ dependent. In conclusion, UNG2 is present in preassembled complexes proficient in BER. Furthermore, UNG2 is the major enzyme initiating BER of deaminated cytosine (U/G), and possibly the sole enzyme initiating BER of misincorporated uracil (U/A)