Noguchi et al.
Supplementary Materials & Methods
page 1
SUPPLEMENTARY MATERIALS AND METHODS
HsOrc1 Cloning and Mutagenesis
A full-length human Orc1 open reading frame (ORF) was cloned from HeLa cell cDNA
library (Invitrogen) by PCR amplification using platinum pfx polymerase (Invitrogen) with primes
(5’-CACAgtcgacggatccATGGCACACTACCCCACAAGGCTGAAG-3’
5’-ACACgcggccgcggatccTTACTCGTCTTTCAGCGCATACAGCAC-3’),
and
and
cloned
into
pCR-TOPO Blunt plasmid vector (Invitrogen). SalI-BamHI and BamHI-NotI sites (lower case)
were added at 5’-end and 3’-end of HsOrc1 ORF respectively. The cloned HsOrc1 cDNA
sequence was identical to NM004153 (National Center for Biotechnology Information).
Deletion mutants of HsOrc1 were generated by PCR amplification using template
pCR-TOPO-HsOrc1 plasmid, as above. Amino acid substitutions and small deletions were
generated
using
QuikChange®
Site-Directed
Mutagenesis
Kit
(Stratagene)
with
pCR-TOPO-HsOrc1 and the primer sets in Table1. DNA sequences of all cloned PCR products
were verified with a ABI PRISM®-Avant 3100 genetic analyser instrument (Applied Biosystems)
using a BigDye® terminator v3.1 cycle sequencing kit (Applied Biosystems).
The N315 cDNA fragment of HsOrc1 was inserted into pCMV2-FLAG plasmid (Eastman
Kodak). This plasmid expresses a N-terminal FLAG-tagged protein driven by CMV promoter in
transient transfection assays.
Plasmids were purified using Qiagen plasmid purification kits (QIAGEN Inc.).
Table I. DNA primers for alterations in HsOrc1 BAH-domain
Primer
5’-DNA sequence-3’
Fw5Nend
cacagtcgacggatccATGGCACACTACCCCACAAGGCTGAAG
Fw169
gggatccTCAGAACTATTTGCGGAGTTGAATAAACCAC
Rev315
gggatccTTAGCGATGTTCAGGTGAAGCCTTCTTGTCATC
Rev3Cend
acacgcggccgcggatccTTACTCGTCTTTCAGCGCATACAGCAC
FwE111K
CGGAAGCCTGGTGCACAGaaaATATTCTGGTATGATTAC
RevE111K
GTAATCATACCAGAATATtttCTGTGCACCAGGCTTCCG
FwD98-107
GTCCGATTCTGTGAAGTCCCTgccggtGCACAGGAAATATTCTGGTATG
RevD98-107
CATACCAGAATATTTCCTGTGCaccggcAGGGACTTCACAGAATCGGAC
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Supplementary Materials & Methods
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Small letters are either additional restriction enzyme sites or mutated sites. [Orc1(wt) =
Fw5Nend+Rev3Cend;
Orc1(BAH)
=
Fw169+Rev3Cend;
Orc1(E111K)
=
FwE111K+RevE111K; Orc1(H) = FwD98-107+RevD98-107; F315 = Fw5Nend+Rev315].
Retrovirus Vector Expression Vector
pOP-AV is a Moloney mouse leukemia virus expression vector that contains a multi-cloning
site
(5’-ACCATGgactacaaggacgacgatgacaagCTCGATGGAGGAtacccctacgacgtgcccgactacgccGGA
GGACTCGAGGAATTCGAACGCGTTGGCGCGGCCGCC-3’)
encoding
FLAG
and
hemaglutinin (HA) epitopes (lower case) followed by an internal ribosomal entry site upstream of
the puromycin-N-acetyl-transferase gene (confers puromycin resistance) (Radichev et al., 2006;
Vassilev et al., 2001). HsOrc1 cDNAs (SalI-NotI fragment) were inserted into XhoI-NotI in
pOP-AV so that HsOrc1 and puromycin-N-acetyl-transferase are expressed from a bi-cistronic
mRNA. Translation of HsOrc1 cDNA inserted at XhoI-NotI sites begins at the Kozak consensus
sequence (first underline) to produce M-(FLAG)-LDGG-(HA)-GGLDGS-HsOrc1. LDGG and
GGLDGS are spacers. FLAG-[HA]3-tagged HsOrc1 was constructed by inserting an additional
DNA
sequence
(CTCGACtacccctacgacgtgcccgactacgccGGAtacccctacgacgtgcccgactacgccCTCGAG)
encoding
two HA epitopes (lower case) at XhoI site in pOP-AV. SalI-NotI fragment from HsOrc1 cDNA
was then inserted at XhoI-NotI sites in this modified vector so that it expressed
M-(FLAG)-LDGG-(HA)-GGLD-(HA)-G-(HA)-LDGS-HsOrc1.
All
constructions
were
confirmed by DNA sequencing.
Stable Cell Lines
293EBNA1 cells, a human kidney epithelial cell line that expresses EBNA1 protein (293c18,
ATCC #CL-10852), were cultured as monolayers in high glucose DMEM (Gibco) supplemented
with 100 units/ml penicillin, 100 µg/ml streptomycin, 5% fetal bovine serum, under 5% CO2 at
37°C. Stable cell lines expressing recombinant FLAG-HA- and FLAG-triple HA-HsOrc1
molecules were established, as previously described (Radichev et al., 2006; Vassilev et al., 2001).
In brief, retroviruses were produced from Phoenix-A cells after Ca++-phosphate transfection with
pOP-AV constructs, and at 72-h post-transfection, viral supernatants were collected for
Noguchi et al.
transduction
Supplementary Materials & Methods
to
293EBNA
cells
page 3
as
described
in
http://www.stanford.edu/group/nolan/protocols/pro_helper_dep.html. Cells expressing ectopic
HsOrc1 protein were selected by their resistance to 1 µg/ml puromycin (Sigma) and routinely
maintained in the presence of puromycin.
Nuclear Extract
Cells (70-90% confluent) were fractionated into cytosol and nuclei as described by (Mendez
and Stillman, 2000), with modifications. Cells were harvested without trypsin treatment, once
washed with PBS, and suspended at 2x107 cells/ml in a hypotonic buffer (HB) [10 mM
Hepes-NaOH pH7.9, 10 mM KCl, 2 mM MgCl2, 0.34 M sucrose, 10% glycerol, 0.1% Triton
X-100] plus a supplement consisting of 1X protein phosphatase inhibitor cocktail I (Sigma), 1 mM
dithiothreitol, 1 mM phenylmethylsulphonylfluoride, 10 µM leupeptin, 10 µM pepstatin A, 10
µg/ml aprotinin, and 25 µM MG132 (Sigma). Cells were incubated for 5 min on ice. Nuclei were
recovered by centrifugation (1,500xg, 5 min, 4°C) and resuspended in nuclear extraction buffer
(NEB) [20 mM Hepes-NaOH (pH7.9), 2 mM MgCl2, 1 mM EGTA, 25% glycerol, 0.1% Triton
X-100] plus the supplement plus 0.1 to 0.5 M NaCl. The volume of buffer at each step was
adjusted to the equivalent of 2x107 cells/ml. Each extraction with NEB was carried out for 5 min
incubation on ice before again recovering nuclei by centrifugation (12,000xg, 5 min, 4°C). The
final pellet was re-suspended with NEB containing 1X Laemmli buffer , incubated at 90°C for 3
min, and sonicated for 1 min to reduce viscosity before fractionating by SDS gel electrophoresis
and detecting proteins by Western immuno-blotting.
Affinity Chromatography For Orc1 Complexes
After removing hypotonic fraction, freshly prepared soluble nuclear extract (4 ml; the
equivalent of 3-5x108 cells) was prepared in nuclear extraction buffer containing 0.35M NaCl
(NEB350). This was incubated with anti-FLAG M2 agarose (Sigma, 20 µL) for 4-5 h at 4°C with
rotation to bind FLAG-HA-tagged HsOrc1. Agarose-beads were washed six times with 500 µL
NEB350 using HandeeTM spin columns (Pierce) at 2,000xg for 1 min at room temperature. FLAG
tagged protein was eluted for 10 min at room temperature with 0.2 mg/ml FLAG peptide (Sigma)
in NEB350 (50 µL/sample) and again collected using spin columns. For SDS-PAGE, each sample
was mixed with 4X Laemmli buffer to give 1X final concentration and immediately incubated at
Noguchi et al.
Supplementary Materials & Methods
page 4
90°C for 3 min. Samples [15 µL for FH-Orc1(wt), and 3 µL for FH-Orc1(BAH)] were
fractionated by electrophoresis at 60 mA constant current in a 4-12% NuPAGE Bis-Tris
polyacrylamide gel with NuPAGE MOPS-SDS running buffer (Invitrogen). Proteins were
visualized with SilverQuestTM staining kit (Invitrogen) according to the manufacture's protocol.
Western Immuno-Blotting
Samples were fractionated by electrophoresis at 60 mA constant in a 4-12% NuPAGE
Bis-Tris polyacrylamide gel with NuPAGE MOPS-SDS running buffer (Invitrogen). Proteins
were transferred from the gel to a PVDF membrane (Immobilon-P, Millipore) by electro-elution
(300 mA constant using XCell IITM blot module (Invitrogen) for 2-4 h in 25 mM Tris, 192 mM
glycine, 20% methanol, 0.01% SDS), and the membranes were then blocked with 5% non-fat dry
milk in PBS-0.05% Tween 20 for 1 h at room temperature with constant agitation. First antibodies
were diluted in blocking buffer [anti-FLAG M2 and anti-HA.11 (0.5 µg/ml); anti-Mcm3 (Abcam,
ab4460, 1:2000); anti-HsOrc2 (BD Pharmingen, 1:3000), anti-HsOrc1 (Santa Cruz
Biotechnology, N-17, 1:100) and anti-CcnA2 (Santa Cruz Biotechnology, H-432, 0.2 µg/ml)]. The
diluted antibodies were incubated with membrane overnight (14-20 h) in cold room with constant
agitation. First antibodies were detected with either anti-mouse IgG (GE Healthcare), anti-rabbit
IgG (GE Healthcare), or anti-goat IgG (Santa Cruz) conjugated to horse radish peroxidase.
Secondary antibodies were diluted to 1:10,000 and incubated with membrane for 1 h at room
temperature with constant agitation. Chemiluminescence was detected using either SuperSignal
West Pico or West Duro Chemiluminescent substrate (Pierce).
Centrifugal Elutriation
Centrifugal elutriation was carried out using an AvantiTM-J-20I centrifuge with JE-5.0
elutriator system (Beckman Coulter). Cells (3-5 x108 cells) were harvested by trypsinization,
re-suspended in 20 ml loading buffer [PBS, 0.05% BSA (Sigma, fraction V), 0.05% Pluronic®
F-68 (Gibco)], and loaded at 1800 rpm, 11 ml/min, 20°C. The rotor speed was kept constant and
100 ml fractions were collected at increasing flow rates (14-36 ml/min) controlled with
Cole-Palmer MasterFlex L/S pump. First 50 ml volume of each 100 ml was used for experiments.
Collected cells were washed once with PBS, and 106 cells from each fraction were used for FACS
analysis.
Noguchi et al.
Supplementary Materials & Methods
page 5
Fluorescence Activated Cell Sorting (FACS)
To analyze cell cycle profile of elutriation fractionated cells, fractionated cells were washed
once in PBS and fixed in 70% ethanol at -20°C for at least 30 min. Cells (106) were then washed
once with PBS, incubated in 0.2 ml of PI solution [0.1% Triton X-100, PBS, 200 µg/ml RNase A
(Sigma), 20 µg/ml propidium iodide (Sigma)] at 37°C for 1 h, and then diluted with 1 ml PBS. To
monitor transfection efficiency, cells that had been co-transfected with pEGFP-C1 (Clontech)
were trypsinized, collected, fixed with 2% formalin for 30 min at room temperature, washed once
with PBS and re-suspended in PBS. To estimate a cell cycle profile of GFP-expressing cells in a
transient transfection assay, cells were fixed with formalin and stained with PI as above. Data
acquisition was performed using a FACSCalibur instrument (Becton Dickenson Biosciences), and
data analysis was performed using CellQuest and ModFit software to calculate a ratio of
GFP-positive cells and a cell cycle profile.
Southern Blotting-Hybridization
DNA samples were electrophoresed in 0.7% agarose gels, denatured in alkaline buffer (0.5
N NaOH, 1.5 M NaCl) for 30 min, and then alkaline transferred to a nylon membrane (Hybond
N+, GE Healthcare) overnight with standard capillary transfer method. Plasmid DNA was
detected by hybridization with random-prime
32
P-dCTP labeled p818 or pEGFP probe in a
PerfectHybTM plus hybridization buffer (Sigma) containing competitive salmon sperm DNA.
Hybridization was performed overnight at 68 °C and signals were analyzed using LAS-3000
phosphoimager instrument.
Protein T1/2
293EBNA cells were seeded in a 6-well plate coated with poly-D lysine (Biocoat, Becton
Dickinson) at 106 cells/2 ml /well. Medium was replaced the following day with
methionine/cystein free DMEM (#21013-024, Gibco) containing 2 mM glutamine (Gibco) and 5%
dialyzed FBS (Gibco). Cells were cultured for 1 h before adjusting medium to 30 µCi/ml, Pro-mix
L-[35S] labeling mix (GE Healthcare). After 2-h labeling, cells were washed twice with
pre-warmed normal growing medium and then cultured in normal growing medium for the
indicated time. Cells were once washed with PBS, trypsinized, and collected by centrifugation
Noguchi et al.
Supplementary Materials & Methods
page 6
(800xg, 5 min). A soluble nuclear extract was prepared in NEB350, precleared with protein
G-agarose (Sigma) for 1 h in the cold room with rotation, and then incubated with anti-FLAG M2
agarose for 4 h to isolate FLAG-tagged protein. Beads were washed eight times with NEB500, and
then FH-Orc1 released by heat denaturing (90°C, 3 min) in a 25 µL of 1X Laemmli buffer.
Proteins were fractionated by SDS-PAGE and the amount of
35
S-Orc1 determined using
LAS-3000 phosphoimager instrument (Fujifilm medical systems).
Immuno-Fluorescence Assays
Cells were grown on Lab-Tek II chamber slides coated with CC2 (Nalgen Nunc), fixed with
2% formalin for at least 7 min at room temperature, and washed once for 5 min with 20 mM
Tris-HCl (pH 7.5) and 150 mM NaCl. Fixed cells were permeabilized for 10 min at room
temperature with either SDS buffer [50 mM HEPES-NaOH (pH 7.9), 0.5% SDS, 0.5% sodium
deoxycholate, 0.1% Triton X-100, 50 mM NaCl, and 5 mM EDTA], or Triton buffer [0.5% Triton
X-100 in PBS]. Alternatively, cells were fixed 10 min in 99.9% methanol. All three methods gave
the same results, although chromosome definition and FH-Orc1 staining were better with the first
two methods. Permeabilized cells were washed twice in PBS and then incubated for 30 min with
antibody dilution buffer [PBS, 1% goat serum, 0.05% Tween 20] to suppress non-specific staining
by the secondary antibody. Slides were incubated overnight with the primary antibody diluted in
“dilution buffer” at 4 °C, then washed in PBS and incubated with 0.5 µg/ml secondary antibody (1
h at room temperature). Slides were washed in PBS, stained with either DAPI or propidium iodide
(1 µg/ml PBS) for 10 min, washed in PBS, then mounted in Vectashield (H-1400, Vector Labs).
Cells were viewed with a Leica DMIRE2 inverted microscope using a 63x oil immersion objective
attached to a confocal laser scanning unit (Leica TCS-SP2). Images were acquired using the Leica
confocal software version 2.5 and merged pictures were processed using Photoshop® 7.0 software
(Adobe). Primary antibodies were mouse monoclonal anti-FLAG M2 (1 µg/ml, Sigma),
anti-HA.11 (1 µg/ml, Covance), anti-HsOrc2 (1:3000, BD Pharmagen), and anti-HsPCNA (1
µg/ml, FL-261, Santa Cruz). Secondary antibodies were goat anti-rabbit or goat anti-mouse IgG
conjugated with either Alexa Fluor® 488 or 568, highly cross-adsorbed (1:2000, Invitrogen).
Plasmid Replication Assays
Noguchi et al.
Supplementary Materials & Methods
page 7
Cells were seeded at 2x106 cells/60 mm dish and then transfected 24 h later using Effectene®
(QIAGEN) with 0.7 µg p818 (Chaudhuri et al., 2001) + 0.3 µg pEGFP-C1 (Clontech) per 6 cm
dish (Fig. 7), or 0.2 µg p818 + 0.1 µg pEGFP + 0.2 µg pN315/well/1.5x105 cells in a 6-well plate
(Fig. 8). Transfection efficiency (% GFP positive cells) was determined by FACS analysis the next
day for each cell line and in every experiment. Average transfection efficiency for 12 experiments
was 26%±15%.
For plasmid maintenance assays (Fig. 7A), cells were harvested at 24-h post-transfection,
and replated in duplicate by serial dilution onto 60 mm dishes. Those containing 1/5400 of cells
were used as non-selected controls to determine plated cell numbers. One day later, 300 µg/ml
hygromycin B was added, and cells were cultured for 2 weeks. Surviving colonies were fixed with
4% formalin, stained with 0.4% crystal violet in methanol, and counted from digital photographs
using ImageJ 1.33u. The fraction of resistant colonies was based on the fraction of cells
transfected, as determined by the number of GFP-positive cells.
For transient DNA replication assays (Figs. 7B & 8), cells were recovered four days
post-transfection, counted, and plasmid DNA was isolated from Hirt lysates (Wysokenski and
Yates, 1989), purified [QIAprep® Spin Miniprep kit (QIAGEN)], and then dissolved in 50 mM
Tris-HCl (pH 8) with volumes adjusted to give the equivalent of 2x106 transfected cells/100 µL.
DNA was digested with 10 Units BamH1±10 Units DpnI. DNA was detected by Southern
blotting-hybridization using appropriate 32P-labeled DNA probes. Results were quantified using a
LAS-3000 phosphorimager. The N-terminal 315 amino acid region of HsOrc1 was cloned using
PCR and inserted into pCMV2-FLAG (Eastman Kodak).
Chromatin Immuno-Precipitation (ChIP) Assays
Cells were transfected with p818 plasmid, selected for growth in 300 µg/ml hygromycin B
for 2 weeks, and expanded for an additional 2 to 3 weeks in the presence of hygromycin. These
limited passaged cells were used for ChIP assay, and cell lysates were prepared freshly for each
experiment, because HsOrc1 protein appeared degradated after freeze-thawing of lysates. Cells
(2x107 cells/sample) were lysed with hypotonic buffer as described above. Nuclei were
re-suspended in 1 ml pre-warmed hypotonic buffer containing 1.85% formalin without
supplements for 5 min at room temperature and then combined with 0.1 ml 1 M glycine. Fixed
nuclei (2x107 nuclei/sample) were collected by brief centrifugation (1,500xg, 5 min), re-suspended
Noguchi et al.
Supplementary Materials & Methods
page 8
in 1 ml 8M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl (pH 7.5), and 5 mM NaCl, sonicated on ice
(power setting 4, three times 90 sec elaspsed time with 3 sec pulse intervals, Ultrasonic processor,
Misonix), and centrifuged (12,000xg, 5 min). Ten µL of each supernatant was separated as a 1 %
input lysate. Clarified lysates were diluted to 1:10 with ChIP dilution buffer (1.1 % Triton X-100,
1.1 mM EDTA, 50 mM Tris-Cl pH 8), and pre-cleared with 50 µL of protein-G agarose/salmon
sperm DNA (50% suspension, Upstate) for 2 h in cold room with rotation. After centrifugation
(1,500xg, 5 min), 5 µg of either anti-HA.11 or mouse IgG was added to each supernanant before it
was incubated overnight (12-14 h) in the cold room. Protein-G agarose beads/salmon sperm DNA
(25 µL/sample) was added and incubated for 2 h in the cold room with rotation to bind the
immuno-complexes. Agarose beads were then washed three times for 5 min with high-salt buffer
[1% Triton X-100, 0.1% SDS, 2 mM EDTA, 50 mM Tris-HCl (pH 8), 0.5 M NaCl), two times for
5 min with LiCl buffer [1% Triton X-100, 0.5% sodium-deoxycholate, 0.25 M LiCl, 1 mM EDTA,
10 mM Tris-HCl (pH 8)] in a 1.5 ml tube, and two times with TE [20 mM Tris-HCl (pH 8), 1 mM
EDTA] in a HandeeTM spin column (Pierce). Immuno-complexes and DNA were eluted twice with
125 µL of elution buffer (1% SDS, 0.1 M NaHCO3, 10 mM dithiothreitol) for 15 min at room
temperature. Formaldehyde crosslinks were reversed in 0.3 M NaCl at 65°C for 4 h, digested with
RNase A (37°C, 30 min), and proteinase K (45°C, overnight). The DNA was purified twice with
phenol/chroloform extraction, and precipitated with ethanol in the presence of glycogen. Input
lysate samples were also treated with same procedure. Purified DNA was redissolved in 100 µL 50
mM Tris-HCl (pH 8).
Quantitative Real Time PCR
Five µL of purified DNA was used as template for real-time PCR reactions performed with
ABI PRISM® 7000 sequence detection system using PowerSYBR® Green PCR Master Mix
(Applied Biosystems) in a 25 µL volume. Amplification efficiency of SC5 and E15 primer sets
was Econst=1.91 and 1.95 respectively, in our reaction condition (10 min at 95 °C, cycle with 15 sec
at 95 °C, 1 min at 60 °C.). PCR reaction was done by triplicate, and purified p818 was used each
time for a standard curve and data calculation.
Noguchi et al.
Supplementary Materials & Methods
page 9
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initiation factors, ORC and MCM, associate with oriP of Epstein-Barr virus. Proc Natl
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Mendez, J. and Stillman, B. (2000) Chromatin association of human origin recognition complex,
cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of
prereplication complexes in late mitosis. Mol Cell Biol, 20, 8602-8612.
Radichev, I., Kwon, S.W., Zhao, Y., DePamphilis, M.L. and Vassilev, A. (2006) Genetic analysis
of human Orc2 reveals specific domains that are required in vivo for assembly and nuclear
localization of the origin recognition complex. J Biol Chem, 281, 23264-23273.
Vassilev, A., Kaneko, K.J., Shu, H., Zhao, Y. and DePamphilis, M.L. (2001) TEAD/TEF
transcription factors utilize the activation domain of YAP65, a Src/Yes-associated protein
localized in the cytoplasm. Genes Dev, 15, 1229-1241.
Wysokenski, D.A. and Yates, J.L. (1989) Multiple EBNA1-binding sites are required to form an
EBNA1-dependent enhancer and to activate a minimal replicative origin within oriP of
Epstein-Barr virus. J Virol, 63, 2657-2666.