Recombinant protein expression and purification
Detailed creation of GST-Ubc13 and GST-Mms2 fusion constructs, their overexpression, protease cleavage, and purification have been described previously (McKenna et al., 2001
). The GST-Uev1A and GST-Uev1B constructs were made by PCR amplification of UEV1A
(obtained from Z.J. Chen, University of Texas Southwestern Medical Center, Dallas, TX) and UEV1B
; obtained from S. Lin, Robert Wood Johnson Medical School, Piscataway, NJ) cDNA clones; the resulting fragments were cloned into pGEX6p (GE Healthcare). The GST-Ubc13K92R construct was created by site-directed mutagenesis and the GST-Uev1AΔ30 construct was made by PCR amplification that removes the NH2
-terminal 30-aa coding region from Uev1A. Each cloned insert was confirmed by DNA sequencing before further analysis. Fusion protein overexpression and purification were performed in a manner similar to that of GST-Ubc13 and GST-Mms2 (McKenna et al., 2001
GST pull-downs were performed using MicroSpin GST Purification Modules (GE Healthcare). 500 μl of bacterial crude cell extract containing GST-Mms2, GST-Uev1A, or GST-Uev1B was loaded and incubated in the purification module for 1 h at 4°C with gentle rocking. The module was then washed three times with 500 μl PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.3); 30 μg of purified Ubc13 in PBS was added and the incubation was continued for another hour at 4°C. The module was washed again three times with 500 μl PBS, and then 80 μl of reduced glutathione buffer (10 mM glutathione in 50 mM Tris-HCl, pH 8.0) was added to elute MicroSpin module-bound proteins. The elution samples were subjected to SDS-PAGE analysis.
Yeast two-hybrid analysis
The hMMS2, UEV1A, and UEV1B coding regions without stop codons were PCR amplified as BamHI–SalI fragments and cloned into pG4BD-1 (received from R.B. Brazas, University of California, San Francisco, San Francisco, CA) as COOH-terminal fusions to Gal4BD. The UBC13-coding region was PCR amplified as an EcoRI–SalI fragment and cloned into pGAD424 (CLONTECH Laboratories, Inc.) as an NH2-terminal fusion to Gal4AD.
Yeast cells were grown in either rich YPD or synthetic SD minimal media at 30°C as described previously (Sherman et al., 1983
) and were transformed with DNA by a LiAc protocol (Ito et al., 1983
). Yeast strain PJ69-4A (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4
Δ LYS2::GAL1-HIS3 GAL2-ADE2 met::GAL7-lacZ
; received from P. James, University of Wisconsin, Madison, WI) was cotransformed with pG4BD-1– and pGAD424-based constructs, and the transformants were selected on SD-Trp-Leu plates. At least five independent transformants were picked from each plate and replicated onto SD-Trp-Leu-Ade to detect activation of the PGAL2
-ADE2 reporter gene or onto SD-Trp-Leu-His supplemented with various concentrations of 3-aminotriazole to measure activation of the PGAL1
-HIS3 reporter gene.
Functional analysis of human genes in yeast
A wild-type haploid S. cerevisiae
strain, HK580-10D (MAT
αade-1 can1-100 his3 11,15 leu2-3, 112 trp1-1 ura3-1
), was received from H. Klein (New York University, New York, NY) and used as the recipient to delete the entire MMS2
open reading frame by a one-step gene replacement method (Rothstein, 1983
) using an mms2
cassette generated through PCR amplification as previously described (Xiao et al., 1999
). The resulting mms2
Δ strain, WXY903, was transformed with two-hybrid plasmids carrying hMMS2
, and UEV1B
The gradient plate assay was performed as previously described (Xiao et al., 2000
) to a semiquantitative measurement of relative MMS sensitivity.
Recombinant human Ubc13 and Mms2 proteins were emulsified in Freund's incomplete adjuvant. Immediately before i.p. injection of BALB/c mice, the emulsion was dispersed with an equal volume of PBS containing 2% Tween 80 (injection volume per mouse of 0.8 ml). Repeat injections of antigen were given at minimum intervals of 3 wk over several months. Fusion cells were screened for secretion of a mAb with reactivity to either Ubc13 or Mms2 using standard enzyme immunoassay techniques in 96-well plates. Hybridomas 4E11 and 2H11 were isolated based on their ability to secrete mAbs recognizing Ubc13 and Mms2, respectively. Hybridoma cells (~106) were injected into the peritoneal cavity of BALB/c mice that had received an i.p. injection of 0.3 ml of Freund's incomplete adjuvant 24 h before. Ascites fluid was collected as the mAb source.
Western blot analysis
Mouse NIH 3T3 cells were grown to log phase and lysed in Dulbecco's PBS (150 mM NaCl, 10 mM Na2HPO4, and 10 mM NaH2PO4, pH 7.4) with 1% SDS and the protease inhibitor cocktail for mammalian cells (Sigma-Aldrich). Total protein concentration was determined by the Bradford method using a commercial reagent from Bio-Rad Laboratories. Cell extracts or purified proteins were electrophoresed in 12% SDS-PAGE gels, transferred to PVDF membrane, incubated with mAb and a biotin-conjugated goat anti–mouse IgG secondary antibody (Sigma-Aldrich), followed by incubation with Streptavidin-HRP and DAB plus hydrogen peroxide for color development.
Constructs to express myc-tagged proteins
MMS2, UEV1A, and UEV1AΔ30 open reading frames without stop codons were PCR-amplified as BamHI–XhoI fragments and then cloned into the BamHI–XhoI sites of pcDNA3.1/Myc-His(+)A (Invitrogen) so that the genes of interest are under the control of a CMV constitutive promoter and fused in frame with the myc-His6 coding region at the COOH terminus.
RNAi and siRNA designs
RNAi constructs were created by cloning double-stranded oligonucleotides at XbaI and BbsI sites of the plasmid vector mU6pro (a gift from D. Turner, University of Michigan, Ann Arbor MI) as described previously (Yu et al., 2002
). Double-stranded siRNAs were synthesized with 3′dTdT overhangs by Dharmacon. They were designed to recognize the target sequences as depicted in Fig. S3.
Cell culture, transfection, and treatments
Human and mouse cell lines were routinely grown in DME (Sigma-Aldrich) containing 4.5 g glucose and 10% horse serum (Invitrogen), with sodium bicarbonate reduced to 2.1 g/liter, in a humidified 5% CO2
incubator. For transfection experiments, 50 μl of serum-free DME containing ~2 μg DNA and 1 μl Lipofectamine 2000 reagent (Invitrogen) was added to log-phase cells grown on 11 × 22-mm coverslips containing 100 μl of complete growth medium. After a 16-h incubation, the coverslips were returned to complete growth media and analyzed as specified. It was estimated that typical transfection efficiency was <10% using pcDNA3.1-derived constructs and >90% using RNAi constructs. For DNA damage treatment, log-phase cells were incubated continuously with CPT, followed by washing and immunocytochemistry (ICC). Microglia were isolated from newborn CD1 mice by aseptically pressing neopalia cleaned of meninges through 70-μm nitex mesh (BD Biosciences) and subsequently cultured in DME (high glucose) plus 10% horse serum as described previously (Hao et al., 1991
). After 12-d culture, they were exposed to LPS and processed for ICC.
For routine ICC, cells were fixed in 4% formaldehyde in Dulbecco's PBS for 30 min, permeabilized by treating with 0.5% Triton X-100 for 5 min, and treated with a blocking solution containing 5% horse serum and 2% skim milk in PBS for 30 min. Primary antibodies used in this study include rabbit anti-Mre11 (1:200; Oncogene Research Products), rabbit anti-Rad51 (1:100; Santa Cruz Biotechnology, Inc.), mAb 2H11 (1:100), mAb 4E11 (1:100), mAb 9E10 (1:400; Sigma-Aldrich), Alexa546-conjugated mouse anti-BrdU (1:400; Molecular Probes), and rabbit anti-myc (1:400; Santa Cruz Biotechnology, Inc.). The green fluorescing Alexa488–conjugated anti–mouse (1:3,000; Molecular Probes) and the red fluorescing Alexa546-conjugated anti–rabbit (1:2,000; Molecular Probes) antibodies were used as secondary antibodies. The secondary antibody solution also contained 2 μg/ml DAPI to visualize the nucleus. Both primary and secondary antibodies were diluted in blocking solution and applied to cells for 30 min, each followed by three rinses with PBS over 30 min. The coverslips were then mounted in PBS and observed using an inverted fluorescence microscope (model IX70; Olympus) fitted with the appropriate filters. Digital images were taken using an RT Slider “Spot” camera and associated software (Diagnostic Instruments). Statistical data were compiled and analyzed using Microsoft Excel and GraphPad QuickCalcs Software (GraphPad Software, Inc.).
To visualize the incorporation of BrdU into DNA, cells were treated with 50 μg/ml DNase-free RNase A immediately after the permeabilization step, and the DNA was then denatured by treating cells with 2 N HCl for 15 min at 65°C before the blocking step. To differentiate the mouse mAbs 4E11 and 2H11 from mouse anti-BrdU, a modified procedure was developed. After the secondary Alexa488 anti–mouse antibody was applied to identify 4E11 and 2H11, coverslips were rinsed thoroughly (six changes of PBS over 1 h). The cells were again blocked by incubating with 2% normal mouse serum for 30 min to obstruct unoccupied anti–mouse Fab regions of bound Alexa488 anti–mouse antibody. Alexa546 anti-BrdU was then applied at a 1:400 dilution for 15 min. To visualize damage-induced nuclear foci (Tomilin et al., 2001
), in situ cell fractionation was performed before fixation by treating cells with 0.4% NP-40 in PBS for 3.5 min with gentle agitation.
NF-κB luciferase reporter and NEMO ubiquitination assays
HEK 293T cells were plated in 6-well plates 18 h before transfection. For siRNA delivery, cells were transfected with 25 nmol of the indicated siRNA, using Lipofectamine 2000 (Invitrogen). 24 h after the first transfection, the cells were transfected again with the same amount of siRNA together with the indicated expression plasmids. Approximately 36 h after the second transfection, the cells were collected and used for different experiments. NF-κB reporter activity was measured using a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Immunoblot analysis of NEMO ubiquitination was performed as described previously (Zhou et al., 2004
In vitro ubiquitination assay
A 0.5-ml conjugation reaction containing 20 nM Uba1, 2.5 μM 35S-labeled Ub, and 250 nM Ubc13 in an ATP cocktail (10 mM Hepes, pH 7.5, 5 mM MgCl2, 5 mM ATP, and 0.6 U/ml inorganic phosphatase) were incubated at 30°C for 90 min. The concentration of each component is noted in the figure legends. Reactions were terminated by the addition of TCA to a final concentration of 10% and processed for a 12% SDS-PAGE analysis by autoradiography.
Image acquisition and processing
All photographic images were taken through a microscope (model IX70; Olympus) with a camera (SPOT RT Slider; Diagnostic Instruments) at RT. Fluorochromes used include Alexa488 (green; Molecular Probes), Alexa546 (red; Molecular Probes), and DAPI (blue; Sigma-Aldrich). For and , an LC PlanFL 40×/0.60 (air) objective (Olympus) was used. For and Fig. S2, an UPlanFLN 60×/1.25 oil immersion objective (Olympus) was used. Images were acquired using Image-Pro Plus version 4.1 software and compiled using Adobe Photoshop version 6. In each plate, photographs were cropped and each Fluorochrome adjusted identically for brightness and contrast to represent the observed images. In , the cyan channel was adjusted identically in all panels to accentuate the merged layer.
Online supplemental material
Fig. S1 shows quantitative analysis of nuclear foci–containing cells after CPT treatment. Fig. S2 shows cellular localization of Mms2-myc and Uev1-myc proteins. Fig. S3 shows RNAi and siRNA sequences. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200502113/DC1