() Recruitment of EGFP-tagged proteins related to DNA repair synthesis to DNA damage induced by a 365-nm UVA-laser

<p><b>Copyright information:</b></p><p>Taken from "Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells"</p><p></p><p>Nucleic Acids Research 2007;35(9):2913-2923.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888830.</p><p>© 2007 The Author(s)</p> (A) Nuclei (arrowheads) of HeLa cells expressing EGFP-RFC140, EGFP-PCNA and EGFP-Polδ1, were irradiated with a 365-nm UVA laser as described in ‘Materials and Methods’ section. () Accumulation of EGFP-fusions, RFC140 (square), PCNA (triangle) and Polδ1 (circle) in (A) was measured as the fold increase of fluorescence intensity at an irradiated site. Data were taken from five independent experiments. Error bars represent standard errors. () Intensity at laser irradiation sites of EGFP-fusions and five RFC subunits was plotted as in (B). () Maximum intensity (MI) and the time to reach MI ( MAX) were represented in each GFP-fusion. A half of MI (1/2 MI) was calculated as 0.5 × (MI − 1) + 1. Thus, 1/2 MI indicates the time to reach 50% of MI

Cellular localization of endogenous RFC140 and PCNA in asynchronous 293T cells

<p><b>Copyright information:</b></p><p>Taken from "Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells"</p><p></p><p>Nucleic Acids Research 2007;35(9):2913-2923.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888830.</p><p>© 2007 The Author(s)</p> S1, S2 and P2 indicate cytoplasmic, nucleoplasm (soluble nuclear) and chromatin/nuclear matrix (insoluble nuclear) fractions, respectively. MEK2 and ORC2 blots are represented as a markers specific to cytoplasmic and chromatin fractions, respectively

Accumulation kinetics of a series of deletion fragments with or without F6A/F7A substitution

<p><b>Copyright information:</b></p><p>Taken from "Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells"</p><p></p><p>Nucleic Acids Research 2007;35(9):2913-2923.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888830.</p><p>© 2007 The Author(s)</p> () Sequence alignment of the N-terminal portion of RFC140 orthologs from nine eukaryotes. Approximately 10 amino-acid residues are indicated. Red-colored residues are highly conserved among all species, and are indicated as the PIP-box (see text). EGFP-fused fragments 1–369 (), 1–397 (), 1–493 (), 1–733 () and full-length (1–1147, ) having normal (closed squares) or the F6A/F7A mutation (open squares) were transiently expressed in HeLa cells and accumulation was measured as the fold increase of fluorescence intensity. Error bars indicate standard error. Error bars not indicated are smaller than symbols. Pictures represent accumulation of FA mutant fragments, and are taken before and 120 min after laser irradiation. Data were taken from five independent experiments. White arrowheads indicate the laser irradiation region

Accumulation of EGFP-RFC140 and EGFP-PCNA in response to laser-induced DNA damage in xrcc1-deficient and xrcc1-proficient mouse embryonic cells

<p><b>Copyright information:</b></p><p>Taken from "Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living human cells"</p><p></p><p>Nucleic Acids Research 2007;35(9):2913-2923.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888830.</p><p>© 2007 The Author(s)</p> Nuclei of xrcc1-deficient (KO) or proficient (WT) mouse embryonic cells expressing EGFP-RFC140 or EGFP-PCNA were irradiated with a low dose (SSBs) or a high dose (SSBs + DSBs) of 365-nm UVA-laser light. Time-lapse pictures were taken as indicated in A

Accumulation of XRCC1 and LIGIIIα at SSBs after local UV-irradiation in XPA-UVDE cells

<p><b>Copyright information:</b></p><p>Taken from "Translocation of XRCC1 and DNA ligase IIIα from centrosomes to chromosomes in response to DNA damage in mitotic human cells"</p><p>Nucleic Acids Research 2005;33(1):422-429.</p><p>Published online 14 Jan 2005</p><p>PMCID:PMC546168.</p><p>© 2005, the authors © </p> Co-localization of XRCC1 with LIGIIIα was identified by double immunolabeling. Two minutes after local UV-irradiation (20 J/m) cells were fixed with paraformaldehyde and co-stained with anti-XRCC1 antibody (red, upper row) and anti-LIGIIIα antibody (green, middle row); the column c is for XPA-UVDE cells treated with DIQ, an inhibitor of PARP, before UV-irradiation. Co-localization of XRCC1 with LIGIIIα appears yellow in overlay (bottom row)

The Role of hnRPUL1 Involved in DNA Damage Response Is Related to PARP1

<div><p>Heterogeneous nuclear ribonucleoprotein U-like 1 (hnRPUL1) -also known as adenovirus early region 1B-associated proteins 5 (E1B-AP5) - plays a role in RNA metabolism. Recently, hnRPUL1 has also been shown to be involved in DNA damage response, but the function of hnRPUL1 in response to DNA damage remains unclear. Here, we have demonstrated that hnRPUL1 is associated with PARP1 and recruited to DNA double-strand breaks (DSBs) sites in a PARP1-mediated poly (ADP-ribosyl) ation dependent manner. In turn, hnRPUL1 knockdown enhances the recruitment of PARP1 to DSBs sites. Specifically, we showed that hnRPUL1 is also implicated in the transcriptional regulation of PARP1 gene. Thus, we propose hnRPUL1 as a new component related to PARP1 in DNA damage response and repair.</p> </div

HnRPUL1 is recruited to laser-irradiated sites via the C-terminus (aa 561–756).

<p>(A) Schematic presentation of hnRPUL1 domains (top) and deletion mutants (left) and the results of recruitment experiments (right). (B) Live cell imaging of irradiated HeLa cells expressing EGFP-tagged deletion mutants of hnRPUL1. Arrows indicate the sites of irradiation.</p

Recruitment of hnRPUL1 is dependent on poly (ADP-ribosyl) ation mediated by PARP1.

<p>(A) Recruitment of hnRPUL1 is inhibited by the poly (ADP-ribose) polymerase inhibitor DIQ in HeLa. (B) EGFP-hnRPUL1 is not recruited to irradiated sites in PARP1-deficient MEF cell, but does in PARP1-proficient WT MEF cell. (C) EGFP-hnRPUL1 is recruited to the site of laser-irradiation by coexpression of the EGFP-tagged PARP1 in PARP1-deficient MEF cell. (D) Immunochemical detection of colocalization of endogenous hnRRPUL1 with PARP1 after laser irradiation with BrdU pre-treatment in HeLa cells. Arrows indicate the sites of irradiation.</p

HnRPUL1 knockdown reduces PARP1 expression by transcriptional regulation.

<p>(A) The level of PARP1 proteins is analyzed by western blotting of total extracts from hnRPUL1 knockdown cell and parallel mock knockdown cell. (B)Analysis of the level of PARP1 proteins after MG132 treatment. (C) The level of PARP1 mRNA is analyzed by quantitative real-time PCR in hnRPUL1 knockdown cell line and a parallel mock knockdown cell with and without MG132 treatment. Error bars represent standard errors from three independent experiments.</p

Recruitment of hnRPUL1 to DSBs sites.

<p>(A) No recruitment of hnRPUL1 to UVDE-induced SSBs sites indicated by CPD in XPA-UVDE cells after local UV irradiation (60 J/m²). (B) Recruitment of EGFP-hnRPUL1 to DSBs sites induced by I-SceI indicated by focus of EGFP-hnRPUL1 colocalized with that of Cherry-tTA-ER after transfection of the plasmid pCMV-NLS-I-SceI (+I-SceI). Arrows indicate the sites of DSBs produced by I-SceI.</p