Depletion of CDT1, p21, and CLASPN partially rescues the hyper DNA damage response in MLN4924 treated cells.

<p>Knockdown efficiency is shown in Figure S8 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101844#pone.0101844.s001" target="_blank">File S1</a>. Knockdown of WEE1 was notably toxic, and only viable cells were counted.</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

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

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

Model.

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

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

Irregular transcription is linked with transcription-replication conflicts in BMI1 and RNF2-deficient cells.

A-B. ChIP using the Rpb1 (p-Ser2) antibody followed by qPCR amplification with the indicated primers demonstrates that the elongation of RNAPII is increased at the tested CFSs in RNF2 knockdown (siRNF2) (A) and KO (B) cells. (N = 3 biological replicates; ***P C. (Left) schematic of primer binding locations within the FRA16D locus (Right) ChIP using a Rpb1 (p-Ser2) antibody followed by qPCR amplification with indicated primers. (N = 3 biological replicates; ***P D. (Left) Representative images demonstrating that the PLA signals between Rpb1 (p-Ser2) and PCNA is increased in T80 cells depleted of RNF2 or BMI1. (Right) Quantification of the percentage of PLA-positive nuclei under the indicated conditions (N = 100 cells per condition from 3 biological replicates). E. The PLA signal between Rpb1 (p-Ser2) and PCNA is restored to normal levels by treatment with the transcriptional inhibitors DRB or α-Amanitin. Quantification of the percentage of PLA positive cells under the indicated conditions (N = 100 nucleus per condition from 3 biological replicates). F. T80 RNF2 KO cells were transfected with 3xFLAG-RNF2 WT, fixed at 36 hours post-transfection, then analyzed for PLA as in D. The assays were done in triplicates (N = 120 for each condition). G. Schematic presentation for detection of collisions between the replisome and RNAPII at nascent replication forks by PLA between biotin-labeled EdU and RNAPII. H. (Left) Representative images demonstrating that PLA signal between Rpb1 (p-Ser2) and EdU-labeled replication forks is increased in U2OS cells depleted of BMI1 and RNF2. (Right) Quantification of the average PLA signals per nucleus and biotin-only control PLA is shown. (N = 50 from 3 biological replicates).</p

R-loops are accumulated at CFSs and replication forks in RNF2-deficient cells.

A. T80 cells were transfected with pyCAG_RNaseH1_ D210N plasmid and subjected to ChIP with the anti-V5 antibody. qPCRs using indicated primers show that the R-loops are enriched at CFSs in the RNF2 KO cells (N = 3 biological replicates; ***P B. ChIP using S9.6 antibody and amplification with the indicated primers by qPCR shows that R-loops are increased at CFSs in RNF2 KO T80 cells (N = 3 biological replicates, ***P C. (Top) ChIP using FANCD2 antibody and amplification with the indicated primers by qPCR shows that FANCD2 is enriched at CFSs in RNF2 KO T80 Cells. (Bottom) Western blot confirming FANCD2 expression and IP efficiency in T80 WT and RNF2 KO cells (N = 3 biological replicates; ***P D. ChIP using FANCD2 antibody and amplification with the indicated primers by qPCR shows that FANCD2 is enriched at CFSs in BMI1 Knockdown T80 Cells. (N = 3 biological replicates; ***P E. ChIP using FANCD2 antibody and amplification with the indicated primers by qPCR shows that FANCD2 enrichment at CFSs in RNF2 KO T80 cells is reduced by expressing exogenous RNH1 WT. There was no significant change upon expressing RNH1 D210N (N = 3 biological replicates). F. (Top) Representative images of FANCD2 and RPA foci in WT and RNF2 KO cells. Where indicated, cells were transfected with pyCAG_RNaseH1_ WT plasmid. (Bottom) Quantification of overlap between the FANCD2 and RPA signals by Pearson’s correlation (N = 50 from 3 biological replicates). G. T80 WT and RNF2 KO cells were crosslinked, and the lysates were subjected to immunoprecipitation with the S9.6 antibody and the eluates were analyzed by western blots for indicated proteins.</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