Cell culture, transfection, and siRNAs
HeLa Tet-On (Invitrogen) cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 10 mM L-glutamine. For cell-cycle arrest in G1/S or mitosis, cells were treated for 24 h with 2 mM thymidine or for 16 h with 100 ng/ml nocodazole, respectively. At 40–50% confluency, cells were transfected with plasmids or siRNAs with Effectene (Qiagen) or Lipofectamine RNAiMax (Invitrogen), respectively, according to the manufacturer's instructions. To establish stable cell lines, HeLa Tet-On cells were transfected with pTRE2-Myc-based plasmids encoding RNAi-resistant Myc-PICH4M (contain silent mutations resistant to siPICH4). Selection of stable clones was performed in the presence of 300 μg/ml hygromycin. The surviving clones were screened for the induced expression of Myc-PICH in the absence or presence of 1 μg/ml doxycycline (Invitrogen).
For RNAi experiments, the siRNAs were chemically synthesized at Dharmacon. The siRNA oligonucleotides used in this study are siPICH4, AGGCCAGACUUAAUGAAAAdTdT; siBLM, GGAGCACAUCUGUAAAUUAdTdT; siTopo IIIα, GAAACUAUCUGGAUGUGUAdTdT; siRMI1, GGCCCAAAUGAAUAAACAAdTdT; and siRMI2, GUAGAAGAUUUACACAGGAdTdT. The siRNAs were transfected at a final concentration of 1 nM.
Antibodies, immunoblotting, and immunoprecipitation
To generate antibodies against PICH, the fragment of PICH (residues 791–1000) was produced in bacteria as a His6
-tagged fusion protein and purified. The protein was used to immunize rabbits at Yenzym Antibodies (South San Francisco, CA). The antisera were purified with Affi-Gel beads (Bio-Rad) coupled to the antigen. The production of anti-APC2 and anti-Mad2 antibodies was described previously (Fang et al, 1998
; Tang et al, 2001
). The commercial antibodies used in this study were as follows: CREST (ImmunoVision), anti-BLAP75/RMI1 (Abcam, ab70525), rabbit anti-BLM (Abcam, ab2179), goat anti-BLM (Santa Cruz, sc-7790), anti-Myc (Roche, 11667203001), anti-Plk1 (Santa Cruz, SC-17783). For immunoblotting and immunofluorescence, the antibodies were used at a final concentration of 1 μg/ml.
For immunoblotting, cells were lysed in SDS sample buffer, sonicated, boiled, separated by SDS–PAGE, and blotted with the indicated antibodies. Horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG (Amersham Biosciences) was used as the secondary antibodies, and immunoblots were developed using the ECL reagent (Amersham Biosciences) according to the manufacturer's protocols and exposed to film.
For immunoprecipitation, whole-cell lysate was prepared by lysing cells in NP-40 lysis buffer (50 mM Tris–HCl pH 7.7, 150 mM NaCl, 0.5% v/v Nonidet P-40, 1 mM DTT and 1 × protease inhibitor cocktail) on ice for 15 min, sonicating three times, then centrifuging the lysed cells at 16 000 g for 15 min at 4 °C. Affinity-purified anti-PICH or anti-Myc coupled to Affi-Prep Protein A beads (Bio-Rad) at a concentration of 1 mg/ml were incubated with the supernatants for 2 h at 4 °C. The beads were then washed five times with the NP-40 lysis buffer. The proteins bound to the beads were dissolved in SDS sample buffer, separated by SDS–PAGE, and blotted with the appropriate antibodies.
Immunofluorescence and live cell imaging
For immunofluorescence, HeLa Tet-On cells or HeLa Tet-On cells expressing H2B-GFP were plated in four-well chamber slides (LabTek) and treated as indicated. Cells were fixed with 4% paraformaldehyde in 250 mM HEPES pH 7.4, 0.1% Triton X-100 at 4 °C for 20 min. After 3–5 washes over 20 min in PBS, cells were permeabilized in 0.5% Triton X-100 for 20 min and then washed with PBS. The cells were blocked in PBS plus 5% fetal bovine serum followed by a 16-h incubation with the primary antibodies. After 3–5 PBS washes over 20 min, cells were incubated with fluorescent secondary antibodies (Alexa Fluor 488 or 647, Molecular Probes) for 30 min at room temperature. After incubation, cells were washed with PBS and their nuclei were stained with DAPI (1 μg/ml). Slides were mounted and viewed with a × 100 objective on a Deltavision microscope. All images were taken at 0.2 μm intervals, deconvolved, and stacked. The images were further processed in ImageJ and pseudo-coloured in Adobe Photoshop. For quantification, multiple random fields were captured and 50–200 cells were counted in each of the three independent experiments.
For live cell imaging, HeLa Tet-On cells stably expressing GFP-tubulin and H2B-mCherry were plated in four-well chamber coverslips (LabTek), transfected with indicated siRNAs against PICH or BLM for 48 h, synchronized at G1/S by thymidine arrest for 18 h and released into fresh medium for 5 h before filming. For visualizing BLM or PICH localization, HeLa Tet-On cells were transfected with plasmids encoding GFP-BLM and mCherry-PICH for 48 h, synchronized at G1/S by thymidine arrest for 18 h and released into fresh medium for 5 h before filming. Cells were imaged in CO2-independent medium (Invitrogen) at 37 °C in a humidified chamber using a Deltavision microscope. Three Z-stacks were acquired every 3 or 5 min for 24 h. Image manipulations (contrast enhancement, cropping, and conversion to QuickTime movies) were performed with ImageJ.
Nucleosome sliding assay
The RSC complex (Rsc2-TAP) and the Chd1-TAP were purified through the tandem-affinity purification approach from budding yeasts (Li et al, 2005
-tagged PICH, PICH K128A, PICH HD (residues 60–680), PICH–BLM, and PICH–BLM helicase-dead mutant were expressed in Sf9 insect cells and purified with the Ni2+
-NTA beads (Qiagen). Mononucleosomes were reconstituted with a 32
P-labelled 216 bp DNA including the 601 sequence through the octamer transfer method (Owen-Hughes et al, 1999
), and gel purified as described previously (Li et al, 2007
). Sliding assays were performed at 37 °C in the sliding buffer (20 mM
HEPES pH 7.9, 50 mM
KCl, 10 mM
, 0.5 mM
PMSF, 2 mM
DTT, 0.05% Igepal CA-830, 10% glycerol, and 100 μg/ml BSA) in the presence of 4 mM
ATP or ATPγS. A removing mix of 750 ng calf thymus DNA and 500 ng oligonucleosomes was added to stop the reactions (Li et al, 2005
). The reaction mixtures were resolved by a native polyacrylamide gel followed by autoradiography.
HeLa Tet-On cells were transfected with the indicated siRNAs for 24 h. The cells were replated and incubated in the presence of 100 μM BrdU for 30 h (two cell divisions). Colcemid (150 ng/ml) was added during the final 30 min to enrich for mitotic cells. Cells were collected by trypsinization and washed in PBS. Cells were swelled in 75 mM KCl for 16 min at 37 °C, followed by centrifugation. Cell pellets were resuspended in fixative (3:1 solution of methanol:glacial acetic acid) and incubated for 20 min at 4 °C. Cells were washed in fixative two more times. After the final wash, cells were resuspended in fixative and dropped onto cold slides. Slides were allowed to air dry in the dark for 2–3 days. Chromosomes were then differentially stained for 5 min with 0.1 mg/ml acridine orange (Molecular Probes). Slides were washed extensively for 2 min under running water followed by a 1-min incubation and mounting in Sorenson Buffer, pH 6.8 (0.1 M Na2HPO4, 0.1 M NaH2PO4). Slides were immediately viewed with a × 63 objective on a Zeiss Axiovert 200 M fluorescence microscope. Images were acquired with a CCD camera using Slidebook imaging software (Intelligent Imaging Innovations). Images were analysed for the number of SCE by counting the number of crossover events per chromosome. About 200 chromosomes were scored for each experiment. Three independent experiments were performed.
Two 50 nt DNA oligonucleotides were designed to form a 25-bp partial duplex when annealed. Before annealing, one oligonucleotide was 5′ 32P-labelled with T4 polynucleotide kinase (Promega) and purified using a Sephadex G50 spin column (Ambion). In all, 10 pmol labelled, purified oligonucleotide was combined with 100 pmol complimentary oligonucleotide in an annealing buffer containing 10 mM Tris–HCl pH 7.4 and 10 mM MgCl2. The duplex substrate was diluted to 10 nM in annealing buffer and stored at −20 °C. PICH, BLM, or the PICH–BLM complex was serially diluted, and added to a helicase buffer containing 20 mM Tris–HCl pH 7.4, 2 mM MgCl2 2 mM ATP, 0.1 mg/ml UltraPure BSA (Ambion), 1 mM DTT, and 1 nM 32P-labelled replication-fork substrate. After a 30-min incubation at 37 °C, TBE DNA loading buffer was added. The samples were separated on a native 15% acrylamide TBE gel (Bio-Rad). The gel was dried and analysed with a phosphoimager.