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Mammalian RAD52 Functions in Break-Induced Replication Repair of Collapsed DNA Replication Forks

Summary Human cancers are characterized by the presence of oncogene-induced DNA replication stress (DRS), making them dependent on repair pathways such as break-induced replication (BIR) for damaged DNA replication forks. To better understand BIR, we performed a targeted siRNA screen for genes whose depletion inhibited G1 to S phase progression when oncogenic cyclin E was overexpressed. RAD52, a gene dispensable for normal development in mice, was among the top hits. In cells in which fork collapse was induced by oncogenes or chemicals, the Rad52 protein localized to DRS foci. Depletion of Rad52 by siRNA or knockout of the gene by CRISPR/Cas9 compromised restart of collapsed forks and led to DNA damage in cells experiencing DRS. Furthermore, in cancer-prone, heterozygous APC mutant mice, homozygous deletion of the Rad52 gene suppressed tumor growth and prolonged lifespan. We therefore propose that mammalian RAD52 facilitates repair of collapsed DNA replication forks in cancer cells.

(B) Sequences of the genomic loci targeted by CRISPR/Cas9 in three different clones of U2OS-Cyclin E cells. Two different mutant alleles were detected in clone 2G, whereas clones 3C and 4A harbored a single mutant allele. In clone 3C, a wild-type RAD52 allele was also detected.
(C) Inactivation of the RAD52 gene has small to modest effects on the static cell cycle profile of U2OS cells inducibly overexpressing cyclin E. The flow cytometry profiles of the three clones, in which the RAD52 gene was targeted by CRISPR/Cas9, were compared to the profile of the parental cells (wt). The cells expressed normal levels of cyclin E (NE) or had cyclin E overexpressed (OE) for four days prior to the flow cytometry analysis. EdU was added to the media 1 hour before harvesting the cells. PI, propidium iodide.
(D) Means and standard deviations of the percentages of cells in the G1, S and G2 phases of the cell cycle, as determined by flow cytometry profiles, like the one shown in panel C (experiment performed in triplicate). One and two asterisks denote statistical significance levels of P<0.05 and P<0.01, respectively, and relevant statistical parameters are listed in Table S6.
(E) Deletion of the RAD52 gene leads to reduced levels of DNA synthesis, as assessed by EdU incorporation. U2OS parental cells (wt) and clone 2G with both alleles of RAD52 inactivated were labeled with EdU for 30 min and then examined by flow cytometry. The cells in G1 and S (colored red in the genomic DNA content versus EdU incorporation plots) were gated and the degree of DNA synthesis was monitored by histogram plots showing the levels of EdU incorporation. The histograms show overall higher levels of EdU incorporation in the parental cells, than in the 2G clone. PI, propidium iodide.
(F) Deletion of the RAD52 gene slows progression through S phase and the cell cycle in general. U2OS parental cells (wt) and clone 2G with both alleles of RAD52 inactivated were pulsed with EdU for 30 min and then cultured for an additional 12 hours, before being examined by flow cytometry. The EdU-positive cells were gated and genomic DNA content was monitored by histograms showing the levels of propidium iodide (PI) staining. The parental cells have higher peaks corresponding to G1 DNA content than clone 2G cells, indicating faster overall progression through the cell cycle. The G2 DNA content peaks of the wt cells are also better defined than the corresponding peaks of clone 2G cells, indicating faster progression through S phase.  (A) Collapse of DNA replication forks after treatment of U2OS cells with HU and a Cdc7 inhibitor for 24 hours. Cells were pulse-labeled with CldU for 40 min, then incubated with HU or HU plus a Cdc7 inhibitor (NMS-1116354) for 6 or 24 hours and finally pulse-labeled with IdU for 40 min in the presence or absence of the Cdc7 inhibitor, as indicated. Fork status was monitored by DNA fiber analysis. Re, restarted forks; Te, terminated forks; NF, newlyfired forks. The Cdc7 inhibitor inhibits new origin firing. This would include origins near collapsed forks, whose firing may be misinterpreted as fork restart.
(D) Rad52 facilitates restart of collapsed DNA replication forks. U2OS parental cells (wt) and the RAD52 knockout (KO) clone 2G were pulse-labeled for 1 hour with CldU followed by a 24 hour treatment with 2 mM HU and 5 µM Cdc7 inhibitor. The cells were then released from the HU block into media containing the Cdc7 inhibitor and IdU for 1 hour. Representative DNA fiber images, like the ones used to calculate the frequencies of fork restart in Fig. 3D are shown. CldU, red; IdU, green. Representative immunohistochemistry (IHC) examples for γH2AX in small (diameter < 1.5 mm), medium (diameter 2-2.5 mm) and large (diameter 3-7 mm) tumors from Rad52 +/+ ;Apc f/+ ;CMVcre and Rad52 -/-;Apc f/+ ;CMVcre mice. Table S1, related to Figure 1A.

siRNAs and plasmids
The Flow cytometry screen U2OS-CycE cells were plated in 6-well plates in the presence or absence of tetracycline and 24 hours later were transfected with siRNAs. The siRNAs were used at a final concentration of 10 to 50 nM and trasfected using either the Hiperfect reagent (Qiagen, 301707) or the Interferin reagent (Polyplus transfection, 409-50) according to the manufacturer's instructions. 72 hours later, the cells were treated for 1 hour with 10 µM EdU, then for 6 hours with 0.1 mM nocodazole and finally with 10 µM BrdU for 1 hour, before being harvested and fixed overnight at 4°C in 90% ice-cold methanol. The flow cytometry staining was performed as previously described (Costantino et al., 2014) with the following modifications: before staining with the anti-BrdU antibody (BD Biosciences, 555627), cells were blocked in PBS containing 1% BSA for 1 hour. Moreover, both the primary and the secondary antibodies were diluted in PBS containing 1% BSA.

Generation of U2OS-Cyclin E-RAD52 knockout cells
The pX458_Rad52_Crispr2 (Guide Sequence Insert: CACCGCCGGAGCTTCCGCTG-GTGCG) construct targeting exon 9 and the pX458_Rad52_Crispr6 construct (Guide Sequence Insert: CACCGTACATAAGTAGCCGCATGGC) targeting exon 3 of the human RAD52 gene were designed and generated as described in the MIT CRISPR tool (Cong et al., 2013). U2OS-CycE cells were transfected with the 2 CRISPR/Cas9 constructs and GFP-positive cells were sorted by FACS in 96-well plates. Single clones were expanded and genomic DNA extracted and used for PCR-based amplification of the targeted loci. For the targeted loci in exons 9 and 3, the CATCCGCCGTGGAGAGCGAGGCC and GGAACTGCTGCTGCAGCTGCTTC or the CCCTGAGGCAGAGGCTGGGCCCAG and CTCCTACCTTCTGGCCTCCGCC primers were used, respectively. Clones that appeared to have PCR products longer or shorter compared to the PCR product from the control wildtype cells were tested for Rad52 expression by western blotting. The genomic PCR products from clones with defective Rad52 expression were cloned into pCR-Blunt II-TOPO vector using the Zero Blunt TOPO PCR Cloning kit (Invitrogen, 450245) and analyzed by Sanger sequencing.

γH2AX detection by flow cytometry
For γH2AX detection by flow cytometry, U2OS cells were seeded in 6-well plates and the next day transfected with the indicated siRNAs at a final concentration of 40 nM using the Interferin reagent. 48 hours after siRNA transfection, the cells were treated with 2 mM HU for 0, 2 or 24 hours. The cells were then fixed with 70% ice-cold ethanol and left overnight at -20°C. The staining was performed using the FlowCellect Histone H2AX Phosphorylation Assay Kit (Millipore, FCCS100182) according to the manufacturer's instructions. The genomic DNA was stained by incubating the cells in PBS containing RNase (Roche, 11119915001) and propidium iodide (PI) (Sigma, P4170).

Immunofluorescence
U2OS-CycE cells were seeded on glass coverslips and 4 days after cyclin E overexpression were fixed in ice-cold methanol for 15 min at -20°C. U2OS cells were also treated for 0, 2 or 24 hours with 2 mM HU or 2 µM CPT and fixed. Cells were permeabilized in PBS containing 0.2% Triton X-100 (Sigma-Aldrich). The permeabilized cells were blocked with PBS containing 1% BSA and then were incubated with the corresponding primary antibodies (listed on the table below) for 2 hours at room temperature followed by 1 hour incubation with the secondary-antibody conjugates Alexa Fluor 488 (Thermo Fischer Scientific, A11001 or A11008) and Alexa Fluor 594 (Thermo Fischer Scientific, A11016). More than 80 cells per replicate and per condition were counted. The threshold to determine if a cell was positive for Rad52 foci was set at 20 foci per nucleus and for Rad51 at 10 foci per nucleus.

Preparation of Chromatin Extracts
U2OS cells were exposed to 2 mM HU for 24 hours or to 9 Gy ionizing radiation 1 hour prior to harvesting. Where indicated, the cells were exposed to HU in the presence of 10 µM ATR inhibitor (VE-821). For subcellular fractionation, the cells were harvested, incubated in Buffer A [8 µM Zinc Acetate, 10 mM Hepes pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 10 mM NaPO4 pH 8.0, and protease inhibitor cocktail (Roche, 05892970001)] for 10 min on ice and then centrifuged for 5 min at 1000 rpm at 4°C. The cells were resuspended in Buffer A, lysed using a Dounce glass homogenizer and centrifuged for 15 min at 3000 rpm at 4°C. The pellets were washed once more in Buffer A and centrifuged for 5 min at 1000 rpm at 4°C. Subsequently, the pellets were resuspended in Buffer B [8 µM Zinc Acetate, 20 mM Hepes pH 7.9, 1.5 mM MgCl 2 , 300 mM KCl, 0.2 mM EDTA pH 8, 10 mM NaPO4 pH 8, and protease inhibitor cocktail], incubated for 1 hour on ice and centrifuged for 15 min at 3000 rpm at 4°C. The pellets, which correspond to the chromatin fraction, were resuspended in sonication buffer [50 mM Hepes pH 7.9, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na deoxycolate, 0.1% SDS, protease inhibitor cocktail] and sonicated in a Bioruptor Pico sonicator (Diagenode). The samples were centrifuged for 15 min at 14000 rpm at 4°C and the supernatant which contained the chromatin fraction was collected and stored at - 20°C. For the phosphatase assay, the samples were treated as before but this time in buffers without EDTA and without phosphatase inhibitor cocktail. The samples were then treated with 800 units of λ-phosphatase, while in the control samples only the λ-phosphatase buffer was added without λ-phosphatase for 30 min at 30°C.

BIR-GFP reporter assay
U2OS cells with a stably integrated reporter construct for monitoring BIR were generated by transfecting the cells with the pBIR-GFP plasmid and then by selecting for stably-transfected clones (Costantino et al., 2014). The clone with the highest level of GFP induction after transfection with a plasmid expressing the I-SceI nuclease was selected for further experiments. In this clone, about 6% of the cells became GFP-positive, when I-SceI was expressed. For depletion of selected proteins, the following siRNAs were used: control, Dharmacon D-001810-10-05; POLD3, Dharmacon L-026692-01-0005; POLD4, Dharmacon L-014013-02-0005; RAD52, Dharmacon L-011760-00-0005. The cells were plated and two days later transfected with 20 ng of the indicated siRNAs and with 3 µg of the plasmid expressing I-SceI (pCMV-3xNLS-I-SceI) by Nucleofection using the Nucleofector Program X-01 (Amaxa-Lonza). Expression of GFP was monitored by flow cytometry 48 hours after transfection with the plasmid expressing I-SceI.

DNA fiber analysis
For the DNA fiber analysis, U2OS cells were pulse-labeled with 40 µM CldU for 40 min and then treated for 6 or 24 hours with 2 mM HU and with or without 5 µM Cdc7 inhibitor. Following the HU treatment, the cells were pulse-labeled with 400 µM IdU for 40 min or 1 hour in the presence or absence of 5 µM Cdc7 inhibitor. DNA fibers were prepared and stained as described previously (Ray Chaudhuri et al., 2012). Briefly, the cells were harvested, lysed and the DNA fibers were spread on APS-coated cover glass (Matsunami Trading, 7017.90000). The DNA fibers were denatured in 2.5 M HCl for 1 hour and blocked with PBS containing 0.2% Tween 20 and 2% BSA. The cover glasses were then incubated with primary antibodies against CldU (Abcam, ab6326) and IdU (BD Biosciences, 347580) for 2.5 hours. For visualization of CldU and IdU, the secondary antibodies Cy3 AffiniPure (Jackson ImmunoResearch, 712-166-153) and Alexa Fluor 488 (Thermo Fischer Scientific, A11001) were used, respectively. Finally, the fluorescence signal was amplified by using the secondary antibodies Dylight 550 conjugate (Thermo Fischer Scientific, SA5-10063) and the Alexa Fluor 488 (Thermo Fischer Scientific, A21467). More than 80 fibers per condition were counted.

Synthesis of the Cdc7 inhibitor
The synthesis strategy of the Cdc7 inhibitor (NMS-1116354; Montagnoli et al., 2010b) required a total of 8 steps including two steps for preparing precursors that are not commercially available (Fig. S5). The main steps were: i) the central aromatic pyrrole formation via a Knorr's reaction, ii) a regioselective electrophilic acylation, iii) the construction of the 2-amino-pyrimidine and iv) the primary amide formation. Except for the Knorr's reaction, which had a moderate yield of 34%, the yields of the other synthetic steps were good and reached at least 77%. The overall yield of the performed synthesis route was 17%. The identity and structural assignments of the intermediates and the final compound were assessed by 1 H-NMR, 13 C-NMR and LRMS. Spectral data were in total agreement with the structural formula of the synthesized compounds. Additionally, the identity and the quality of the final Cdc7 inhibitor were assessed by HRMS (HRMS (ESI+): expected m/z. 348.0413 for C 15 H 11 N 5 OCl 2 [M+H]+; Found m/z: 348.0408) and High Performance Liquid Chromatography (purity >95%).