Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official VSports app下载. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. V体育官网.

. 2014 Jul 15;9(7):e101844.
doi: 10.1371/journal.pone.0101844. eCollection 2014.

Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases

Scott Cukras  1 , Nicholas Morffy  1 , Takbum Ohn  2 , Younghoon Kee  1
Affiliations

Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases (V体育官网入口)

VSports在线直播 - Scott Cukras et al. PLoS One. .

Abstract

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle VSports手机版. .

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Disruption of genomic integrity upon UBE2M silencing.
A. γ-H2AX foci was measured upon expression of shRNA targeting UBE2M 3′UTR, then rescued by expressing siRNA-resistant UBE2M WT or C111S mutant. The western blot analysis shows the knockdown efficiency and the expression of FLAG-HA tagged UBE2M WT and C11S mutant. (∼3 kDa shift is predicted). B. Neutral comet assay. HEY cells were transfected with control or two independent UBE2M siRNAs for ∼72 hours before harvest for the analysis. The tail moment is the length of the tail times the density of the tail. % tail DNA is the density of the tail divided by the density of the tail plus the density of the head.
Figure 2
Figure 2. DNA damage response is perturbed by UBE2M silencing.
A. Growth suppression by UBE2M silencing is enhanced by DNA damaging agents. Growth sensitivity of HEY cells in the presence of CPT and PARP inhibitor ABT888 (Figure S2 in File S1) was monitored using clonogenic assay. B. Treatment of HeLa cells with MLN4924 (0.3uM) leads to elevated BRCA1 and RAD51 foci formation. *indicates neddylated form. C. Time course study of RAD51 foci recruitment and resolution upon MLN4924 treatment. D. Time course study of RAD51 foci in UBE2M knockdown cells. E. Cells depleted of UBE2M were analyzed for HR (E) and NHEJ repair (F) assays.
Figure 3
Figure 3. Effects of individual Cullin silencing in genome integrity.
A. γ-H2AX foci induction was measured in HEY cells treated siRNAs against indicated cullins. Knockdown efficiency is shown in right. B. Formation of double strand breaks were measured using neutral comet assay, in HEY cells treated with siRNAs against indicated cullins.
Figure 4
Figure 4. Inhibiting CUL2 neddylation leads to impaired G1-S transition.
A. Double thymidine block experiments were performed in HEY cells treated with DMSO control or MLN4924 and UBE2M siRNA. See Experimental procedure for detailed protocol. The red line was established by selecting the peak value of the cells in G1 (2N) for the control siRNA sample at the zero hour time point. The red line was then kept constant between samples to provide a means of comparison. B. Double thymidine block experiments were performed in HEY cells individually knockdown with indicated cullins. C. Expression of siRNA-resistant CUL2 wild type (WT), but not the empty vector (EV) nor the CUL2 mutant (C689R; ΔNedd8 in the figure), partially rescues the G1-S arrest phenotype. CUL2 siRNA #3 targets the 3′UTR of the CUL2 mRNA. Western blot confirms the knockdown efficiency and ectopic expression of CUL2 proteins. D. Double thymidine block experiments were performed using the HCT116 wild type or p21-/- cells that are treated with either control or CUL2 siRNAs. E. Induction rate of RAD51 foci was measured in HEY cells treated with control or CUL2 siRNAs. The counting was normalized to the 0 time point to indicate the fold increase.
Figure 5
Figure 5. Silencing of CUL4 leads to G2-M checkpoint activation that is associated with DNA repair defects.
A. Resolution of RAD51 foci was measured upon knockdown of individual Cullins. Schematic of the experiment shown in left. B. RAD51 foci kinetics was performed in cells in which CDT2 was stably knockdown. C. Prior depletion of CDT1 or p21 by siRNAs partially rescues the hype-RAD51 foci formation in MLN4924 treated cells. D. Clonogenic assays were performed for the HEY cells knockdown with CUL4A or CDT2. E. HR repair assays F. NHEJ assay.
Figure 6
Figure 6. Depletion of CDT1, p21, and CLASPN partially rescues the hyper DNA damage response in MLN4924 treated cells.
Knockdown efficiency is shown in Figure S8 in File S1. Knockdown of WEE1 was notably toxic, and only viable cells were counted.
Figure 7
Figure 7. Model.
UBE2M inhibition impacts DNA damage response and genome integrity involving multiple Cullin ligases.
See this image and copyright information in PMC

"VSports app下载" References

    1. Huang DT, Ayrault O, Hunt HW, Taherbhoy AM, Duda DM, et al. (2009) E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification. Mol Cell 33: 483–495. - PMC - PubMed
    1. Ma T, Chen Y, Zhang F, Yang CY, Wang S, et al. (2013) RNF111-dependent neddylation activates DNA damage-induced ubiquitination. Mol Cell 49: 897–907. - PMC - PubMed
    1. Xirodimas DP, Saville MK, Bourdon JC, Hay RT, Lane DP (2004) Mdm2-mediated NEDD8 conjugation of p53 inhibits its transcriptional activity. Cell 118: 83–97. - "VSports" PubMed
    1. Duda DM, Scott DC, Calabrese MF, Zimmerman ES, Zheng N, et al. (2011) Structural regulation of cullin-RING ubiquitin ligase complexes. Curr Opin Struct Biol 21: 257–264. - PMC - PubMed
    1. Deshaies RJ, Emberley ED, Saha A (2010) Control of cullin-ring ubiquitin ligase activity by nedd8. Subcell Biochem 54: 41–56. - PubMed

Publication types

MeSH terms