14 research outputs found
Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks
During the DNA damage response (DDR), ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5), a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB) repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair. © 2014 Nakajima et al
Ubiquitin-specific protease 5 is required for the efficient repair of DNA doublestrand breaks. PLoS One. 2014; 9:e84899. [PubMed: 24454762
Abstract During the DNA damage response (DDR), ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5), a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB) repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of cH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair
Altered nucleotide misinsertion fidelity associated with polι-dependent replication at the end of a DNA template
A hallmark of human DNA polymerase ι (polι) is the asymmetric fidelity of replication at template A and T when the enzyme extends primers annealed to a single-stranded template. Here, we report on the efficiency and accuracy of polι-dependent replication at a nick, a gap, the very end of a template and from a mispaired primer. Polι cannot initiate synthesis on a nicked DNA substrate, but fills short gaps efficiently. Surprisingly, polι’s ability to blunt-end a 1 bp recessed terminus is dependent upon the template nucleotide encountered and is highly erroneous. At template G, both C and T are inserted with roughly equal efficiency, whilst at template C, C and A are misinserted 8- and 3-fold more often than the correct base, G. Using substrates containing mispaired primer termini, we show that polι can extend all 12 mispairs, but with differing efficiencies. Polι can also extend a tandem mispair, especially when it is located within a short gap. The enzymatic properties of polι appear consistent with that of a somatic hypermutase and suggest that polι may be one of the low-fidelity DNA polymerases hypothesized to participate in the hypermutation of immunoglobulin variable genes in vivo
The Cullin 4B–Based UV-Damaged DNA-Binding Protein Ligase Binds to UV-Damaged Chromatin and Ubiquitinates Histone H2A
Relationship of the Xeroderma Pigmentosum Group E DNA Repair Defect to the Chromatin and DNA Binding Proteins UV-DDB and Replication Protein A
Cells from complementation groups A through G of the heritable sun-sensitive disorder xeroderma pigmentosum (XP) show defects in nucleotide excision repair of damaged DNA. Proteins representing groups A, B, C, D, F, and G are subunits of the core recognition and incision machinery of repair. XP group E (XP-E) is the mildest form of the disorder, and cells generally show about 50% of the normal repair level. We investigated two protein factors previously implicated in the XP-E defect, UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA). Three newly identified XP-E cell lines (XP23PV, XP25PV, and a line formerly classified as an XP variant) were defective in UV-DDB binding activity but had levels of RPA in the normal range. The XP-E cell extracts did not display a significant nucleotide excision repair defect in vitro, with either UV-irradiated DNA or a uniquely placed cisplatin lesion used as a substrate. Purified UV-DDB protein did not stimulate repair of naked DNA by DDB(−) XP-E cell extracts, but microinjection of the protein into DDB(−) XP-E cells could partially correct the repair defect. RPA stimulated repair in normal, XP-E, or complemented extracts from other XP groups, and so the effect of RPA was not specific for XP-E cell extracts. These data strengthen the connection between XP-E and UV-DDB. Coupled with previous results, the findings suggest that UV-DDB has a role in the repair of DNA in chromatin
USP5 is necessary for cell survival after DNA damage.
<p>A: Knockdown of USP5 expression by siUSP5 treatment. <b>B</b>: Colony forming assay after treatment with Bleocin, hydroxyurea, or methyl-methanesulfonate, with or without siUSP5 treatment. Filled square indicates without siUSP5 treatment and filled circle indicates with siUSP5 treatment; error bars, ± SED. The <i>P</i>-value was calculated using Student's <i>t</i>-test.</p
USP5 co-localizes with DNA DSBs.
<p>A: EGFP-tagged USP5 was expressed in HeLa cells, and cells were irradiated with the laser at a dose of 100 frames with 100 µM of 8-MOP. 8-MOP sensitizes laser light to produce DNA base damage and strand breaks. <b>B</b>: EGFP-tagged USP5 co-localizes with DNA DSBs produced by a restriction enzyme. Plasmid DNAs for expression of USP5-EGFP and Cherry-TA were introduced in U2OS SceI cells with or without NLS-SceI expression plasmid DNA by Lipofect amine 2000. After overnight incubation, cells were fixed and stained by anti-γH2AX antibody. <b>C</b>: Endogenous USP5 co-localizes with DNA DSBs produced by a restriction enzyme. Plasmid DNA for expression of NLS-SceI was introduced in U2OS SceI cells by Lipofect amine 2000. After overnight incubation, cells were fixed and stained by anti-USP5 antibody and anti-γH2AX antibody. At least 150 cells were counted in each sample. <b>D</b>: Damage response of USP5-EGFP after laser micro-irradiation with or without ATM inhibitor. Cells were irradiated with the laser at a dose of 100 frames with 100 µM of 8-MOP and with or without 10 µM of ATM inhibitor. The results are averages obtained from two independent experiments, and more than 52 cells were irradiated and analyzed for the damage response. <b>E</b>: Domain analysis of USP5 with regard to the damage response. Schematic presentation of domains in USP5 and the GFP-tagged mutant constructs. zf-UBP, Zinc-finger in ubiquitin-hydrolases and other proteins; UBA, Ubiquitin Associated domain. EGFP-tagged USP5 full length or deletion mutant was expressed in HeLa cells, and cells were irradiated with the laser at a dose of 100 frames with 100 µM of 8-MOP. The results are averages obtained from at least two independent experiments and more than 33 cells were irradiated and analyzed.</p
USP5 is required for disassembly of polyubiquitin chains at sites of DNA damage.
<p>A: EGFP-tagged ubiquitin was expressed in HeLa cells with or without siUSP5 treatment. Cells were irradiated with the laser light for 100 ms. The intensity of EGFP-Ub at the irradiated site was analyzed and summarized in the graph. The results are averages obtained from three independent experiments (n = 5). <b>B</b>: Cells were irradiated with the laser for 10 frames with 100 µM of 8-MOP and then fixed and stained by anti-ubiquitin antibody (FK1; left panel, FK2; right panel) and anti-RAD18 antibody. Anti-RAD18 antibody is used for showing irradiated sites.</p
USP5 interacts with RAD18 and USP5 depends on RAD18 in DSB repair.
<p>A: Interaction between expressed proteins. We transiently expressed EGFP-tagged RAD18 in cells expressing FLAG-His-tagged USP5 and pulled down by the His-tag and pull downs were detected with anti-GFP antibody. <b>B</b>: Interaction between EGFP-tagged RAD18 and endogenous USP5. Cells were treated with or without Bleocin for 2 hr and then cells were extracted. Extracts were pulled down with anti-GFP antibody and detected by anti-USP5 antibody. <b>C</b>: Damage response of USP5-EGFP after laser micro-irradiation in RAD18-proficient or -deficient cells. EGFP-tagged USP5 and DsRed-tagged RAD18 WT or each mutant were co-expressed in RAD18-deficient cells, and the damage response after laser micro-irradiation was analyzed. Cells were irradiated with the laser light for 100 ms. <b>D</b>: HR frequencies in cells depleted of USP5 and/or RAD18. Results of western blot analysis after siRNA treatment are shown on the top. The GFP-positive cell fraction in cells depleted of USP5 and/or RAD18 was determined and compared with that in cells treated with siCont or siBRCA1 for determination of frequencies; error bars, ± SED. The <i>P</i>-value was calculated using Student's <i>t</i>-test.</p