Inhibiting CUL2 neddylation leads to impaired G1-S transition.

<p><b>A</b>. 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>B</b>. Double thymidine block experiments were performed in HEY cells individually knockdown with indicated cullins. <b>C</b>. 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. <b>D</b>. Double thymidine block experiments were performed using the HCT116 wild type or p21-/- cells that are treated with either control or CUL2 siRNAs. <b>E</b>. Induction rate of RAD51 foci was measured in HEY cells treated with control or CUL2 siRNAs. The counting was <i>normalized</i> to the 0 time point to indicate the fold increase.</p

DNA damage response is perturbed by UBE2M silencing.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101844#pone.0101844.s001" target="_blank">File S1</a>) was monitored using clonogenic assay. <b>B</b>. Treatment of HeLa cells with MLN4924 (0.3uM) leads to elevated BRCA1 and RAD51 foci formation. *indicates neddylated form. <b>C</b>. Time course study of RAD51 foci recruitment and resolution upon MLN4924 treatment. D. Time course study of RAD51 foci in UBE2M knockdown cells. <b>E</b>. Cells depleted of UBE2M were analyzed for HR (<b>E</b>) and NHEJ repair (F) assays.</p

Model.

<p>UBE2M inhibition impacts DNA damage response and genome integrity involving multiple Cullin ligases.</p

Effects of individual Cullin silencing in genome integrity.

<p><b>A</b>. γ-H2AX foci induction was measured in HEY cells treated siRNAs against indicated cullins. Knockdown efficiency is shown in right. <b>B</b>. Formation of double strand breaks were measured using neutral comet assay, in HEY cells treated with siRNAs against indicated cullins.</p

Disruption of genomic integrity upon UBE2M silencing.

<p><b>A</b>. 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>B</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.</p

Silencing of CUL4 leads to G2-M checkpoint activation that is associated with DNA repair defects.

<p><b>A</b>. Resolution of RAD51 foci was measured upon knockdown of individual Cullins. Schematic of the experiment shown in left. <b>B</b>. RAD51 foci kinetics was performed in cells in which CDT2 was stably knockdown. <b>C</b>. Prior depletion of CDT1 or p21 by siRNAs partially rescues the hype-RAD51 foci formation in MLN4924 treated cells. <b>D</b>. Clonogenic assays were performed for the HEY cells knockdown with CUL4A or CDT2. <b>E</b>. HR repair assays <b>F</b>. NHEJ assay.</p

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair

<p>USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.</p