The Carboxyl Terminus of Brca2 Links the Disassembly of Rad51 Complexes to Mitotic Entry

Summary Background The Rad51 recombinase assembles on DNA to execute homologous DNA recombination (HR). This process is essential to repair replication-associated genomic lesions before cells enter mitosis, but how it is started and stopped during the cell cycle remains poorly understood. Rad51 assembly is regulated by the breast cancer suppressor Brca2, via its evolutionarily conserved BRC repeats, and a distinct carboxy (C)-terminal motif whose biological function is uncertain. Using “hit-and-run” gene targeting to insert single-codon substitutions into the avian Brca2 locus, we report here a previously unrecognized role for the C-terminal motif. Results We show that the avian C-terminal motif is functionally cognate with its human counterpart and identify point mutations that either abolish or enhance Rad51 binding. When these mutations are introduced into Brca2, we find that they affect neither the assembly of Rad51 into nuclear foci on damaged DNA nor DNA repair by HR. Instead, foci disassemble more rapidly in a point mutant that fails to bind Rad51, associated with faster mitotic entry. Conversely, the slower disassembly of foci in a point mutant that constitutively binds Rad51 correlates with delayed mitosis. Indeed, Rad51 foci do not persist in mitotic cells even after G2 checkpoint suppression, suggesting that their disassembly is a prerequisite for chromosome segregation. Conclusions We conclude that Rad51 binding by the C-terminal Brca2 motif is dispensable for the execution of HR but instead links the disassembly of Rad51 complexes to mitotic entry. This mechanism may ensure that HR terminates before chromosome segregation. Our findings assign a biological function for the C-terminal Brca2 motif in a mechanism that coordinates DNA repair with the cell cycle.

successful targeting events was with SacI digestion of PCR products generated by primers PF9 and PR9. The resistance cassettes were then floxed out by transient expression of Cre recombinase. Briefly, cells were transfected with pPGK-Cre plasmid (kind gift from Dr. K.J. Patel) using solution from Nucleofector Kit T according to the Amaxa guidelines. Clones with restored sensitivity to Bsr and Neo were selected. Direct sequencing of RT-PCR product of the observed clone was used to confirm S3232A mutation.
To generate a Brca2 +/cell line, wild type DT40 cells were sequentially transfected with pLoxBsr-KI 5'B2 and pLoxNeo-KI 3'B2 targeting vectors. Southern blotting combined with PFGE analysis was used to confirm the targeting of loxP sites at both the 5' and 3' ends of the same allele of Brca2. For Southern blotting genomic DNA was digested with PshAI and probed with a 32 P-labeled PCR fragment generated by PF10 and PR10 primers. The floxed Brca2 allele was deleted by expressing Cre-recombinase as described above. Southern blotting was carried out on DNA extracted from clones with regained sensitivity to Bsr and Neo. The extracted DNA was digested with either EcorV or BsrGI and hybridized with a 5'-probe generated by PCR using primers PF11 and PR11 or with a 3'-probe generated using primers PF12 and PR12.
The Brca2 P3240L/cell line was generated by transfecting a pLoxBsr-KI P3240L targeting construct into the Brca2 +/cell line. Targeting events were validated as described for the S3239A mutant above. The Brca2 T3232A/mutant cell line was generated using pLoxBsr-KI T3232A and validated as described above except that the PCR product was digested with BseRI instead of SacI.

Immunofluorescence and Antibodies
Immunofluorescence analyses were done as described previously [1]. Briefly, approximately 10 5 cells were spun out onto glass slides in a Shandon Cytospin-2 cytocentrifuge for 5 min at 800 rpm slides were dried at 37°C for 5 minutes and fixed in 4% paraformaldehyde for 15 min at room temperature. Subsequently, slides were then rinsed in PBS and kept at -20°C in 70% EtOH until further analysis. Cells were blocked with 3% BSA in 1XPBS, 0.01% Tween-20, 0.01%Triton and stained with primary antibodies followed by Alexa Fluor 488 or Alexa Fluor 568 secondary antibodies (Molecular Probes, Inc.) The following primary antibodies were used: anti-RAD51 polyclonal Ab (pAb) was purchased from Merck Biosciences, anti-γ-H2AX Ser139 monoclonal antibody (mAb) from Upstate Cell Signalling Solutions (NY, USA), anti-myc mAb (9E10) from Santa Cruz biotechnology, Inc. anti-cyclinB pAb (kind gift from E. A. Nigg), anti-GgBrca2 pAb (kind gift from Professor Shunichi Takeda, Japan). All Western blotting or immunofluorescence reagents were used at the dilutions recommended by the manufacturers. Imaging was performed on a Zeiss LSM510 Meta confocal microscope, using a 40x objective.

Quantitative Immunofluorescence Microscopy
Cells cytospun onto glass slides were stained with rabbit polyclonal anti-Rad51, detected using an Alexa Fluor 488-conjugated anti-rabbit antibody. Slides were mounted with Vectashield medium containing DAPI. Ten-15 representative fields were acquired using a 40x objective on a Zeiss LSM 510 Meta confocal microscope for each sample; using constant zoom and imaging parameters (laser intensities and detector settings). The collected images were batch exported as channel specific TIF files, and renamed (using r-nameit) into the Cellomics data format. The images were imported into a Cellomics HCS arrayscan VTI (ThermoFisher) under the diskscan mode; and analysed using a protocol based on the 'Compartmental analysis' bio-application. Briefly, the nuclear (DAPI) stain was used to identify objects for analysis, and intra-object changes in intensity in the Rad51 channel used to define foci. Typically, 50-500 cells were analysed in each sample to determine parameters including average number of foci per cell and number of cells with more than a defined foci count. Cells containing one or more Rad51 foci were counted as positive to ensure that cells exhibiting a complete dissolution (absence) of foci were stringently enumerated. The data were exported in Excel format and plotted in Graphpad Prism v4.0.
Slides of U2OS cells stained for cyclin B were processed by automated microscopy on an Olympus ScanR High content screening microscope using a 40x non-immersion lens. Slides were autofocused using a stepped image intensity based algorithm for the DAPI nuclear signal, and then 25 40x fields (typically containing 1000-2000 cells) for each slide were imaged. Images of DAPI and TRITC channels were obtained for each position using real time controlled channel specific illumination from a MT20 Hg-Xenon light source, with fixed exposure times between samples. The images were stored in the ScanR format for quantitative analysis. For analysis, a constant background subtraction of 200 arbitrary units (12bit) was used across all channels. The nucleus was defined as the primary object using DAPI intensity, and using the 'watershed' function to separate closely spaced cells. The mean intensity for nuclear cyclin B staining was determined, and cells staining two standard deviations above the population mean were scored as positive.

Sister Chromatid Exchange Assay
The sister chromatid exchange assay was performed as described previously [2]. Briefly, cells were cultured for two cell cycles in the presence of 10µM BrdU and with or without 50ng/ml mitomycin C (MMC) for the second cell cycle. Cells were pulsed with 100ng/ml colcemid for three hours before harvest. Cells were hypertonically swollen in 75mM KCl for 20 minutes at room temperature before fixation in freshly prepared Carnoy's solution (methanol: acetic acid, 3:1) for 30 minutes at room temperature. Fixed cells were dropped onto slides, dried at 50 o C for 20 minutes and then incubated for 20 minutes in 10µg/ml Hoescht-33258 (Sigma), diluted in 0.05M sodium phosphate buffer, pH 6.8 at room temperature. Slides were irradiated with UV-A (365nm) for 90 minutes and then incubated in pre-warmed 2X SSC for 1 hour at 62 o C. Slides were stained with freshly prepared Leishman's stain (Sigma) (diluted 1:3 in 0.05M sodium phosphate buffer, pH 6.8) for 2 minutes. Slides were dried and mounted with Eukitt Mounting Media (Electron Microscopy Sciences) under a coverslip. Cells in metaphase were visualised with a 100X objective on a Zeiss Axioskop 2 microscope and 50 spreads scored blind to the observer.

Immunoglobulin Gene Diversification Assay
The immunoglobulin gene diversification assay was performed as described previously [3]. Briefly, cells were stained with FITC-conjugated goat anti-chicken sIgM (Bethyl Laboratories) for flow cytometric analysis. Single cell sorting of sIgM + subclones was carried out with a Mo-Flo cell sorter (DakoCytomation) and single cells were plated to 96-well plates. 48 subclones per cell line were grown in 24-well plates and split daily. After a four week expansion, subclones were re-stained for sIgM to assess the frequency of generation of sIgM-loss variants. sIgMcells from 12 subclones were collected using a Mo-Flo sorter and subjected to genomic DNA extraction. The VL1 sequence was then amplified by 35 cycles of PCR with CVLF5 and CVLR3 primers and cloned into pBluescript KS+ by SacI (in CVLF5) and HindIII (in CVLR3) restriction sites and transformed into subcloning efficiency DH5α competent cells (Invitrogen). Sequencing by M13F and M13R was carried out from bacterial stab cultures by Lark Technologies and MRC Geneservice and Monte-Carlo sequence analysis was performed with GAP v4.10 (Staden Package) and the MUTHR program based on an algorithm to check novel sequences against the pseudo V-genes in DT40.

Cell-Cycle Analysis and MPM2 Staining
FACS analysis was performed as previously described [4]. For MPM2 analysis, DT40 cells were washed once in PBS, fixed in cold 70% ethanol for 2 hrs, washed with PBS and stained with anti-MPM2 antibody for 1 hr at 37°C (1:1200; Upstate Cell Signalling Solutions NY, USA). Cells were then washed with 1XPBS before staining with Alexa Fluor 488 conjugated secondary antibody (1:500) for 1 hr at room temperature. Cells were stained with propidium iodide before FACS analysis. Mitotic cells were identified as those positive for MPM2 staining.

Cell Survival Assay
Cells were plated into 96-well plates at a density of 8000 cells per well. Different doses of mitomycin C (Sigma), and camptothecin (Sigma) were added, and the plates were incubated at 39°C for five doubling times. In the case of ionizing radiation, cells were exposed to different doses of IR before initial seeding. CellTiter-Blue reagents (Promega) were added to each well of the 96-well plate according to the manufacturer's guidelines. The plates were incubated at 37°C for approximately 1-2 hour in a humidified 5% CO 2 atmosphere. Number of viable cells was determined using the Fusion plate reader at A590 nm. Each experiment was done in quadruplet.

Pulsed-Field Gel Electrophoresis (PFGE) and Southern Blotting
PFGE was carried out using CHEF DR-II PFGE apparatus (BioRad) under the following conditions: 1% agarose gel in 1X TAE buffer, 200 volts at 14°C with an initial switching time of 1 s to a final switching time of 6 s for 48 hrs. For Southern blot, digested DNA samples were separated by electrophoresis and blotted on Hybond-N + membrane (Amersham-Pharmacia) and hybridised with probe labeled with 32-P dCTP (Amersham) by a random priming reaction [5]. After overnight hybridisation and subsequent washing, the radioactive filter was exposed to a Kodak XAR film at -80°C.

Western Blots and Immunoprecipitation
Western blot and immunoprecipitation were performed as described previously [6]. Whole-cell extracts were made in NP-40 lysis buffer. myc-tagged B2-9 was expressed in 293T cells and immunoprecipitated with an anti-myc monoclonal antibody. Immunoprecipitates were washed extensively in NP-40 lysis buffer and resolved on 4-12% gradient Bis-Tris gels (Invitrogen) followed by Western blotting.

In Vitro Kinase Assay
Wild-type, T3232A, S3239A or the double-mutant versions of GST fused to GgBrca2 (3217aa-3252aa) were in vitro phosphorylated in CDK1 reaction buffer, supplemented with 200 μM ATP, 5μCi of [γ-32 P]ATP and 20 U of recombinant CDK1 (New England Biolabs) for 30 min at 30° C. Before reaction termination with SDS sample buffer, the peptides were cleaved from the GST tag using thrombin protease (Amersham) at 25°C for 2 hr. Reaction products were resolved on a 10-20% Tricine gel (Invitrogen) according to the manufacturer's instructions. The ~5 KDa peptides were detected by silver staining (Sigma). Dried gels were exposed to a phosphorimager screen and visualized on a FujiFilm FLA-5000 processor. (B) Confirmation of S3239A knock-in by genomic PCR (using PF9 and PR9 primers) and enzymatic digestion. Successful targeting of the first allele is indicated by the presence of 1.8kb and 0.8kb bands after SacI digestion in addition to the 2.6kb band from the untargeted wild-type allele. Homozygous knock-in is confirmed by complete loss of the 2.6kb band. M indicates 1Kb DNA ladder.
(C) Western blot for GgBrca2 in WT and S3239A mutant cell lines shows comparable protein levels.  (B) Southern blot confirming successful deletion of one allele of Brca2 to generate a heterozygous Brca2 cell line. Presence of the targeting cassettes on the 5' and the 3' end of the GgBrca2 gene is indicated by a 6.7kb and 5.9kb fragment respectively, in addition to the 3.7kb fragment reflecting the presence of the wild-type allele (compare the first two lanes of each blot). Southern blotting confirms the removal of the floxed region ('F' indicates 'floxed') after Cre-mediated recombination, as indicated by the disappearance of the 6.7kb and 5.9kb products compared to the unfloxed heterozygous cell line (the last lane of each blot). The 3'-probe and 5'-probe denote radiolabelled DNA fragments used for Southern blotting (for details, see Experimental Procedures and Figure S2A).       PF1  AGGTCGACAAGTGGTCCACCCCAACTAAAGACTG  PF2  ACGCGTCGACGAATGCTCTTCTCCTTCCTGCAACTCA  PF3  GCGTGAATGCGGCCGCTAGATGCACATGTACAGTACTGTGTA  PF4  CGCGGTACCTTCAGTGGCAGTCTGAATTCAGATCAGA  PF5  ATAGGTACCGTGTCAGCCGGTCACCTCCTTCTGG  PF6  ATACTCGAGGCATGCTGCTGGCTCATGGCCAACC  PF7  CGCGGTACCATCGAAGATATCGTTGTCCGCTGTT  PF8  GCTCTAGATGTGCTGTCTTAGTGTGCATGT  PF9  GTGATCCTGAGTATCTGGCGTCCAC  PF10  CAATAGTTGCACCTGCACAGCCTTCT  PF11  GTGATCCTGAGTATCTGGCGTCCAC  PF12  ACGCGTCGACAAAGCAGTGGACTTCCTCAGCTGCATAC  PR1  GGCCGCGGCCGCATATTTTTTAGTTGTAATTGTGTCCTGC  PR2  ATAAGAATGCGGCCGCTTTGCGGGATTTGCGCCTTTGCAGC  PR3  CGAGCTCGCAATGGCCTTAACATAGCAGGAAGTC  PR4  ACGCGTCGACCTAGTAGCATTTGCGGGATTTGCGCCTTTG  PR5  TATCGTCGACCTGTGGCCAAGGCCAATGGTATCC  PR6  ATCAGCATGCGGCCGCAGTTGATGCCAACTGGCTGTCCGTAG  PR7  ACGCGTCGACACATGCACACTAAGACAGCACATT  PR8  CTGAGCTCGGTACAGAACTGTGCCAGAATTACAGAGG  PR9  GCCACAAGATTATAGCTTTCATCAG  PR10  TCAGCGCCGAACTCACGAACTGAGC  PR11  GCCACAAGATTATAGCTTTCATCAG  PR12  ATAAGAATGCGGCCGCCAAACGTCGCGGAGGCTGAAATGC  TG-SacI  CCTCTCCATTTAGCTTTGAGCTCAGACACAGCGTAAATG  AT-BseRI  CATACCTGCCCCTCCTCCACTGGCACCGCTCTGTTCCATCATTT  CVLF5  CAGGAGCTCGGCTCTGTCCCATTGCTGCGCGG  CVLR3 GCGCAAGCTTCCCCAGCCTGCCGCCAAGTCCAAG