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. 2015 Jan;43(2):987-99.
doi: 10.1093/nar/gku1384. Epub 2015 Jan 7.

"V体育官网入口" Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice

Sonia Jimeno  1 , María Jesús Fernández-Ávila  2 , Andrés Cruz-García  1 , Cristina Cepeda-García  2 , Daniel Gómez-Cabello  2 , Pablo Huertas  3
Affiliations

Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice (V体育2025版)

Sonia Jimeno et al. Nucleic Acids Res. 2015 Jan.

Abstract

DNA double strand breaks are the most cytotoxic lesions that can occur on the DNA. They can be repaired by different mechanisms and optimal survival requires a tight control between them. Here we uncover protein deneddylation as a major controller of repair pathway choice VSports手机版. Neddylation inhibition changes the normal repair profile toward an increase on homologous recombination. Indeed, RNF111/UBE2M-mediated neddylation acts as an inhibitor of BRCA1 and CtIP-mediated DNA end resection, a key process in repair pathway choice. By controlling the length of ssDNA produced during DNA resection, protein neddylation not only affects the choice between NHEJ and homologous recombination but also controls the balance between different recombination subpathways. Thus, protein neddylation status has a great impact in the way cells respond to DNA breaks. .

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Figures

Figure 1.
Figure 1.
Protein neddylation inhibits CtIP-mediated DNA-end resection and HR. (A) Schematic representation of the SeeSaw 2.0 reporter. A GFP gene is flanked by two truncated parts of RFP gene (RF and FP) sharing 302 bp of homologous sequence. Two I-SceI-target sites were cloned at the 3′end of the GFP gene in opposite orientation. After generation of a DSB by I-SceI expression, the damage may be resolved by NHEJ, thus cells will express the GFP protein, or using the homologous sequence by HR, creating a functional RFP gene. (B) Effect of different inhibitors in the SSR 2.0. To measure the deviation from the balance between NHEJ and HR, the ratio between green versus red cells in each conditions was calculated. To facilitate the comparison between experiments, this ratio was normalized with control cells treated with DMSO. Those conditions that skew the balance toward an increase NHEJ result in fold increase above 1. On the contrary, a net increase of this ratio (values below 1) represents an imbalance of the SSR toward HR. Data represent a minimum of three sets of duplicated experiments. (C) DNA-end resection efficiency measured as the percentage of cells positive for RPA foci. Cells expressing a control shRNA (shScr) or an shRNA against CtIP (shCtIP) were pretreated with 0.2 μM of MLN4924 (MLN) or DMSO for 1 h, then irradiated (10 Gy) and incubated for an additional hour in the presence of the inhibitor. Bars represent the average and standard deviation of three independent experiments. A representative image of each case is shown. (D) As in (C), but cells transfected with a plasmid bearing an HA-NEDD8 gene or HA as a control. Asterisk represent statistical significance as described in the Methods section.
Figure 2.
Figure 2.
MLN4924 phenotypes in HR/NHEJ balance and DNA resection depend on CtIP. (A) Cells containing an shRNA against CtIP or a control shRNA (shScr) were transfected with an shRNA resistant GFP-CtIP fusion. One hour after MLN4924 or DMSO addition, cells were irradiated and an additional hour later the amount of cells showing RPA foci was scored. Other details are the same as in Figure 1C. (B) Cells expressing an shRNA against CtIP or control were treated for 12 days with MLN4924 0,1 μM or DMSO. The number of colonies formed in the presence of MLN4924 was normalized with the number of colonies in the DMSO control and plotted. Bars represent the average and standard deviation of three independent experiments. (C) Cells expressing the indicated shRNAs were irradiated with 2 Gy, with and without 1-h preincubation with 0.2 μM of MLN4924 or DMSO, then incubated 2 h with the inhibitor and then for 12 days in fresh medium. The number of colonies formed normalized with a control not irradiated is shown. Bars represent the average and standard deviation of three independent experiments. (D) The ratio between HR and NHEJ was calculated with the SSR system in cells expressing the indicated shRNAs and treated with different doses of MLN4924 as indicated. Details are the same as in (B). Statistical significance was calculated with a 2-way ANOVA.
Figure 3.
Figure 3.
CtIP and BRCA1 complex formation is controlled by their interaction with neddylated proteins. (A) CtIP and BRCA interact with neddylated proteins. Protein extracts were divided into two and immunoprecipitated with an anti-NEDD8 antibody or a non-related IgG as a control and blotted with the indicated antibodies. A representative experiment is shown. (B) Protein samples from cells pre-treated with MLN2449 or DMSO were immunoprecipitated with either an anti-NEDD8 antibody or mix of two anti-BRCA1 antibodies and blotted with anti-CtIP or anti-BRCA1. A representative western blot is shown. (C) The amount of immunoprecipitated protein with anti-NEDD8 antibody from (B) was relativized to the input and then normalized to DMSO, taken as 100%. The average and standard deviation from three independent experiments is shown. (D) Cells were irradiated with 10 Gy and protein samples collected at the indicated time points. After immunoprecipitation with an anti-NEDD8 antibody, samples were blotted for CtIP and BRCA1. A representative western blot is shown. (E) Quantification of panel (D). The quantification of the amount of immunoprecipitated protein relative to the input and normalized to time 0 from three independent experiments is shown. (F) Quantification of BRCA1 IP from panel (B). Details are the same as in (C). (G) Average number of RIF1 foci per cells. Cells pre-treated with either DMSO or MLN4924 were immunostained with a RIF1 antibody. The graph represents the average and standard deviation of the number of foci per cell from three independent experiments. A representative image of each case is shown. (H) Same as (G), but cells transfected with HA-NEDD8 or an empty plasmid. The average of six independent experiments is shown.
Figure 4.
Figure 4.
Role of RNF111/UBE2M in DNA end resection and DSB repair pathway choice. (A) Cells depleted for RNF111 or control cells were analyzed for RPA foci formation as described in Figure 1C. A representative immunofluorescence is shown. (B) Same as in (A), but cells were analyzed for RIF1 foci as described in the Materials and Methods section. (C) The balance between HR and NHEJ was calculated with the SSR system in cells transfected with siRNAs against the indicated genes or control siRNA (siScr). Other details are the same as in Figure 1B. (D) Same as in (C), but cells depleted for UBE2M. (E) Same as in (A), but cells depleted for UBE2M. (F) As in (B), but cells depleted for UBE2M. (G) Protein samples from cells previously downregulated for UBE2M were immunoprecipitated with a mix of two anti-BRCA1 antibodies and blotted with anti-CtIP or anti-BRCA1. A representative western blot and the quantification from five independent experiments, made as described in Figure 3, are shown.
Figure 5.
Figure 5.
Effect of inhibition of protein neddylation in different DSB repair pathways. (A) In the EJ5 reporter (left), I-SceI-induced DSB can be repaired by NHEJ recreating an active GFP gene, containing or not a functional I-SceI target site. The percentage of green cells was calculated as described in the Materials and Methods section in cells pretreated with MLN4924 or DMSO. This percentage was normalized with the DMSO-treated cells value and plotted. Bars represent the average and standard deviation of three independent experiments. (B) Same as (A), but using the SA-GFP. Such a reporter is formed by two truncated copies of the GFP that, upon SSA, can restore an active GFP gene with the deletion of one of the repeats and the intervening region. Other details are the same as in (A). (C) The DR-GFP reporter is formed by two non-functional copies of the GFP. Gene conversion induced by an I-SceI-mediated DSB restores an active GFP gene. The efficiency of gene conversion was calculated as described in (A) for NHEJ. (D) Single molecule analysis of resection tracks (SMART) of cells treated with DMSO or MLN4924. The length of individual fibers is shown as a scatter plot. A Mann–Whitney test was performed to analyze the statistical difference of both populations. (E) The median length of resected DNA was normalized to DMSO. The average of four independent experiments is shown.
Figure 6.
Figure 6.
The extent of resection influences the outcome of recombination. (A) Neddylation acts as a molecular timer for DNA end resection and repair pathway choice. First, high local protein neddylation due to RNF111 activity inhibits resection favoring NHEJ. After that, NEDP1 and CSN activation reduce protein neddylation, allowing some resection to take place. Most breaks will be repaired by short-track gene conversion. Finally, local protein neddylation is sufficiently reduced to allow hyper-resection, favoring repair such as SSA that requires long tracks of resected DNA. (B) A broken DNA that is going to be engaged in recombination is resected (a) before the 3′ OH overhand is used to invade (b) a homologous region (red lines) located elsewhere (gray lines). DNA synthesis (dashed lines) will use the homologous DNA as a template (c) and it could continue until the end of the chromosome (d; BIR), the newly synthesized DNA can reanneal (e; SDSA recombination subpathway) or it can be ligated with the resected DNA to form two Holliday Junctions (f; DSBR recombination subpathway). Depending on how such structures are resolved, recombination will lead or not to crossing overs (g). However, hyper-resection (right) could lead to the exposure of ssDNA regions that are no longer homolog (h; black versus gray lines). In such scenario, although DNA invasion (i) and DNA synthesis (j–k) might happen, a non-homolog DNA would be used as a template for DNA synthesis (k; gray dashed lines). Thus, the newly synthesized DNA can no longer reanneal with the acceptor DNA (black solid line), effectively blocking SDSA and HJ formation. If additional repeated sequences are located nearby (green lines), the break can be sealed using additional recombination pathways such as SSA (l). An alternative would be that DNA synthesis continues until the end of the chromosome (m; BIR).
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References

    1. Aguilera A., Gomez-Gonzalez B. Genome instability: a mechanistic view of its causes and consequences. Nat. Rev. Genet. 2008;9:204–217. - PubMed
    1. Ciccia A., Elledge S.J. The DNA damage response: making it safe to play with knives. Mol. Cell. 2010;40:179–204. - PMC - PubMed
    1. Jackson S.P., Bartek J. The DNA-damage response in human biology and disease. Nature. 2009;461:1071–1078. - PMC - PubMed
    1. Jackson S.P., Durocher D. Regulation of DNA damage responses by ubiquitin and SUMO. Mol. Cell. 2013;49:795–807. - PubMed (V体育平台登录)
    1. Huertas P. DNA resection in eukaryotes: deciding how to fix the break. Nat. Struct. Mol. Biol. 2010;17:11–16. - PMC - PubMed

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