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.
| Original language | English |
|---|---|
| Article number | e101844 |
| Journal | PLoS ONE |
| Volume | 9 |
| Issue number | 7 |
| DOIs | |
| State | Published - 15 Jul 2014 |
Bibliographical note
Funding Information:We thank Drs. Meera Nanjundan, Min Huang, and Martin Cohn for critical reading of the manuscript. We thank Dr. Bert Vogelstein for kind sharing of the p21 knockout and wild type HCT116 cells. We thank Drs. Maria Jasin and Jeremy Stark for the HR and NHEJ reporter cell line, respectively. We thank Robert Buzzeo for assistance with confocal microscopy, Charlie Szekeres for flow cytometer, and Milad Davini for western blotting. pcDNA-Myc-CUL2 plasmid was a gift of Dr. Yu Xiong (Addgene plasmid #19892). This work was supported, in part, by the University of South Florida Research & Innovation Internal Awards Program under Grant No. 0088622, and USF Proposal Enhancement Grant to Y.K.