From the Department of Genetics, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710
Received for publication, December 21, 2000, and in revised form, January 22, 2001
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ABSTRACT |
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The minichromosome maintenance (MCM) proteins,
together with the origin recognition complex (ORC) proteins and Cdc6,
play an essential role in eukaryotic DNA replication through the
formation of a pre-replication complex at origins of replication. We
used a yeast two-hybrid screen to identify MCM2-interacting proteins. One of the proteins we identified is identical to the ORC1-interacting protein termed HBO1. HBO1 belongs to the MYST family, characterized by
a highly conserved C2HC zinc finger and a putative
histone acetyltransferase domain. Biochemical studies confirmed the
interaction between MCM2 and HBO1 in vitro and in
vivo. An N-terminal domain of MCM2 is necessary for binding to
HBO1, and a C2HC zinc finger of HBO1 is essential for
binding to MCM2. A reverse yeast two-hybrid selection was performed to
isolate an allele of MCM2 that is defective for interaction with HBO1;
this allele was then used to isolate a suppressor mutant of HBO1 that
restores the interaction with the mutant MCM2. This suppressor mutation
was located in the HBO1 zinc finger. Taken together, these findings
strongly suggest that the interaction between MCM2 and HBO1 is direct
and mediated by the C2HC zinc finger of HBO1. The
biochemical and genetic interactions of MYST family protein HBO1 with
two components of the replication apparatus, MCM2 and ORC1, suggest
that HBO1-associated HAT activity may play a direct role in the process
of DNA replication.
Eukaryotic DNA replication is a tightly regulated process that is
strictly coupled to cell cycle progression, ensuring that DNA is
replicated only during S phase and that each origin is used only once
per cell cycle. This precise cell cycle coordination is the result of
both positive and negative regulation of replication origin function.
Genetic and biochemical studies in yeast and metazoans suggest that the
initiation of DNA synthesis is a complex, multistep process that
requires the participation of many proteins (1-3). This process
involves the binding of the origin recognition complex (ORC)1 to
replication origins (4, 5), the recruitment of Cdc6 and the six MCM
proteins (MCM2-7; MCM for minichromosome
maintenance) to form the pre-replicative complex (pre-RC)
(6, 7), and the activation of the pre-RC by protein kinases to initiate
DNA synthesis (8).
MCM proteins were revealed to be involved in DNA replication as the
result of genetic screens for mutants defective in progression through
the cell division cycle (9-12) or the replication of minichromosomes (13-15). Initial characterization of three genes, mcm2
(14), mcm3 (16), and mcm5/cdc46 (9, 17),
implicated each in DNA replication and showed they were related in
sequence. This family rapidly grew to encompass the
Schizosaccharomyces pombe mcm4/cdc21+ (18, 19)
and mcm6/mis5+ genes (15), and the
Saccharomyces cerevisiae mcm7/cdc47 gene (20). Analysis of
the complete S. cerevisiae genome sequence indicates that
there are six MCM-encoding genes, and homologs of the MCM2-7 proteins
have since been identified in all eukaryotes from yeast to humans (for
reviews see Refs. 21-23).
MCM proteins play a critical role in the function and regulation of DNA
replication. A loss-of-function mutation in any mcm gene
impairs replication initiation in S. pombe, indicating that each MCM protein has a unique and essential function in DNA replication (24, 25). Additionally, in mammalian cells microinjection of anti-MCM2
antibodies (26), anti-MCM3 antibodies (27), or antisense oligomers
against MCM7 mRNA (28) blocks DNA replication. In a Xenopus
laevis egg extract in vitro DNA replication system, immunodepletion of MCM3 protein prevents DNA synthesis (29, 30). In
Drosophila, mutations in the mcm2 or
dpa/mcm4 genes inhibit cell proliferation and are homozygous
lethal, with mutants showing prolonged S phases (31, 32). Although MCM
proteins are essential for normal DNA replication, it is not yet clear to what extent MCMs have a unique role in determining replication competence of chromosomal DNA or whether additional factors are involved in this process.
In a search for novel MCM-interacting factors, we identified a protein
that interacts with the MCM2 subunit of the MCM complex. This protein
was initially described as interacting with another component of the
pre-RC, the ORC1 protein, and thus termed Histone acetyltransferase Binding to ORC1 (HBO1) (33).
HBO1 shares sequence similarity with the MYST subfamily of histone
acetyltransferases. MYST subfamily members include the human proteins
MOZ (monocytic leukemia zinc finger protein)
(34), MORF (MOZ-related factor) (35), and Tip60 (HIV Tat-interacting
protein of 60 kDa) (36), Drosophila
MOF (males-absent on the first)
(37), and yeast proteins SAS2 and SAS3 (something
about silencing) (38, 39), and Esa1 (essential SAS family
acetyltransferase) (40). MYST subfamily proteins are
characterized by a highly conserved ~270 amino acid domain that
consists of an atypical C2HC zinc finger motif and a
putative histone acetyltransferase (HAT) domain.
In this study, we demonstrate that MCM2 interacts specifically with
HBO1 both in vitro and in vivo and that this
interaction is conserved in metazoans. Furthermore, we map the
interacting domains of both MCM2 and HBO1 and describe a role for the
C2HC zinc finger of HBO1 in this interaction. We also
identify an interaction-defective allele of MCM2, as well as a
compensating second site suppressor mutation in HBO1 that restores the
interaction with the defective MCM2. These results indicate that two
components of the pre-RC, MCM2 and ORC1, interact with a putative
histone acetyltransferase and suggest a direct role for HAT activity in
the process of DNA replication.
Identification of a cDNA Encoding a Mouse MCM2-binding
Protein--
Full-length mouse MCM2 cDNA with an N-terminal triple
HA tag was excised from pREC-HA3mMCM2 and cloned into the
Asp718 and SpeI sites of the pGBT9B vector
(pGBT9B is a version of the pGBT9 vector (CLONTECH,
Palo Alto, CA) with a modified
polylinker).2 The resulting
vector, pGBT9B-HA3mMCM2 (GBD-mMCM2), contains the TRP1 marker gene and a GAL4-(1-147) DNA-binding domain
(GBD)-HA3MCM2 fusion gene. Yeast strain HF7c (MATa,
ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4-542,
gal80-538,
LYS2::GAL1UAS-GAL1TATA-HIS3, URA3::GAL417-mer(×3)CYC1TATA-lacZ)
carrying the GBD-mMCM2 plasmid was transformed with a human placenta
MatchMaker cDNA library cloned into the pACT2 vector
(CLONTECH), carrying the LEU2 gene and
the GAL4-(768-881) activation domain (GAD). Greater than 2 × 106 transformants were initially screened by histidine
nutritional selection and Reverse Two-hybrid Selection--
PCRs for mutagenesis of amino
acids 72-223 of dMCM2 contained 20 ng of pGBT9B-dMCM2
(GBD-dMCM2-(1-888)), 30 pmol each of primers TB63
(5'-GAGATCCTGGAGACCAGAC-3'), and TB64 (5'-GAATCTCTGTGGTGACACG-3'), 125 µM dNTPs, 50 mM KCl, 10 mM Tris
(pH 9), 0.1% Triton X-100, 1.5 mM MgCl2,
BSA (1 µg/µl), and 5 units of Taq DNA polymerase in 100 µl (1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C for
40 cycles). MnCl2 was added to 100 µM after
10 cycles. The region of dMCM2 corresponding to amino acids 72-234 was
removed from plasmid GBD-dMCM2 by digestion at unique XcmI
and EcoNI sites located at nucleotides 216 and 700 of dMCM2,
respectively, and linearized plasmid DNA was gel-purified (QIAquick,
Qiagen, Valencia, CA). The mutagenized PCR fragments and linearized
expression vector containing homologous sequences were then
introduced directly into yeast strain MAV103 (MATa, leu2-3,
112, trp1-901, his3-200, ade2-101, gal4 Second Site Suppressor Screen--
PCR-mediated mutagenesis of
HBO1-(218-611) was performed as above using primers TB86 (pACT2
upstream; 5'-CCATACGATGTTCCAGATTACGC-3') and TB87 (pACT2
downstream; 5'-GAAATTGAGATGGTGCACGATGC-3') and template
GAD-HBO1-(218-611). MnCl2 was added to 10 µM
after 10 cycles. The region of HBO1 corresponding to amino acids
218-599 was removed from plasmid GAD-HBO1-(218-611) by digestion at
BamHI sites located in the 5' polylinker and at nucleotide
1786 of the HBO1 cDNA, respectively, and the linearized plasmid DNA
was gel-purified (QIAquick, Qiagen). The mutagenized PCR fragments and
linearized expression vector containing homologous sequences were then
introduced directly into yeast strain Y190 (MATa, ura3-52,
his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4 Construction of Plasmid DNAs--
Full-length HBO1 cDNA was
constructed in multiple steps. An SphI-HindIII
HBO1 cDNA fragment was excised from clone TB305 and inserted into
the corresponding sites of pUC119 to generate
pUC119-HBO1(SphI-HindIII). The 5' region of HBO1
was amplified from a human placental cDNA library (MatchMaker;
CLONTECH) by PCR using gene-specific primers TB27
(5'-GATCTCTAGACATATGCCGCGAAGGAAGAGGAATGCAG-3'; introduces XbaI and NdeI sites at 5' end of cDNA) and
TB28 (5'-CTCTCCGTTGGTGCTGGTGCCTG-3'). This PCR fragment was digested
with XbaI and SphI, and subcloned into the same
sites of pUC119-HBO1(SphI-HindIII) to generate
pUC119-HBO1. The nucleotide sequence of the PCR-amplified region of
HBO1 was verified by sequencing. The HBO1 cDNA was subsequently
excised using XbaI and AvrII and subcloned into
the XbaI site of pBlueScript II SK+ (Stratagene,
La Jolla, CA) in both orientations, creating pBS-HBO1(fwd and rev).
GST fusions vectors pGEX-dMCM2, -mMCM2, -dORC1, and -hORC1 were
constructed as follows. Plasmid pGEX-dMCM2 was constructed by
subcloning an Asp718-EcoRI fragment of
Drosophila MCM2 cDNA (from pBS-dMCM2; kindly provided by
Jessica Treisman, New York University Medical Center, New York)
in-frame into the same sites of pGEX-TB1 (pGEX-3X with modified
polylinker).2 pGEX-mMCM2 was cloned as a
BamHI-EcoRI fragment from
pREC-HA3mMCM2 into the corresponding sites of pGEX-2T (AP
Biotech, Piscataway, NJ), with a single HA epitope intact between the
GST and mMCM2 cDNAs. pGEX-dORC1 was constructed in multiple steps.
A MunI-PacI (Klenow-blunted) dORC1 cDNA
fragment from pNB40-dORC1 (43) was blunt-end ligated into the
PstI (T4-blunted) site of pSP73 (Promega). A
SalI-HindIII (Klenow-blunted) dORC1 fragment was
then excised from pSP73-dORC1 and blunt-end ligated into the
EcoRI (Klenow-blunted) site of pGEX-3X (AP Biotech) to
generate pGEX-dORC1. A HindIII to XbaI (both
Klenow-blunted) fragment of human ORC1 cDNA from pcDNA3-hORC1
(44) was ligated in-frame into the BamHI (Klenow-blunted) site of pGEX-2T (AP Biotech) to generate pGEX-hORC1.
Plasmid pGBT9B-dMCM2 (GBD-dMCM2-(1-888)) was constructed by subcloning
Asp718 to PstI fragment of Drosophila
MCM2 cDNA (from pBS-dMCM2) in-frame into the same sites of pGBT9B.
Plasmids GBD-dMCM2-(466-888) and GBD-dMCM2-(589-888) were generated
by removing NcoI-NcoI fragments (either nt
1-1444 or 1-1813 of dMCM2 cDNA, respectively) from pGBT9B-dMCM2.
The following C-terminal truncated GBD-dMCM2 fusion proteins were
generated by blunt insertion of an NheI oligonucleotide linker (5'-CTAGCTAGCTAG-3'; New England Biolabs, Beverly, MA) encoding
Amber stop codons in three reading frames into unique restriction sites
(indicated) of pGBT9B-dMCM2: GBD-dMCM2-(1-861) (BglII),
GBD-dMCM2-(1-668) (NdeI), GBD-dMCM2-(1-467)
(NarI), GBD-dMCM2-(1-234) (EcoNI; then remove
NheI to BglII (Klenow-blunted) and used to construct the following three plasmids): GBD-dMCM2-(1-223)
(XcmI), GBD-dMCM2-(1-105) (SalI),
GBD-dMCM2-(1-72) (MscI). GBD-dMCM2-(133-234) was generated
by removal of the XmaI-XmaI fragment from
GBD-dMCM2-(1-234). GBD-dMCM2-(133-234) was constructed by insertion
of XcmI (T4-blunted)-NheI fragment from
GBD-dMCM2-(1-223) into SmaI-NheI sites of
GBD-dMCM2-(1-223). GBD-dMCM2-(1-223) was created by insertion of
NheI linker (see above) into SmaI site of
GBD-dMCM2-(72-223). N-terminal-truncated plasmids GBD-dMCM2-(73-888),
-dMCM2-(106-888), -dMCM2-(133-888), and -dMCM2-(224-888) were
created by replacing an N-terminal fragment of GBD-dMCM2 with the
appropriate PCR-generated fragment. PCR products were digested with
EcoRI (appended to upstream primer) and EcoNI (nt
750 of dMCM2) and inserted into EcoRI (upstream polylinker)
to EcoNI (nt 750) sites of GBD-dMCM2-(1-888) (details available upon request). The DNA sequence of all PCR-generated constructs was verified by sequencing.
All GAD fusion plasmids are constructed in the vector pACT2
(CLONTECH) and have an HA epitope tag at the N
terminus of the inserted cDNA. GAD-HBO1-(218-611) was isolated as
clone TB305 in the mouse MCM2 two-hybrid screen and is in the
EcoRI to XhoI sites of pACT2. Plasmids
GAD-HBO1-(218-542), -HBO1-(218-470), -HBO1-(218-398),
-HBO1-(218-367), -HBO1-(260-611), -HBO1-(320-611), -HBO1-(363-542),
and -HBO1-(363-470) were generated by PCR amplification using
HBO1-specific primers (with BamHI (upstream) and
XhoI (downstream) sites appended) encoding the appropriate
sequences (details available upon request). PCR products were digested
with BamHI and XhoI and inserted into the same
sites of pACT2. Plasmid GAD-HBO1-(384-611) was constructed by removal
of the XmaI (Klenow-blunted) to NaeI fragment of
GAD-HBO1-(218-611). Plasmid GAD-HBO1-(541-611) was constructed by
removal of the XmaI (Klenow-blunted) to BsaBI
fragment of GAD-HBO1-(218-611). GAD-HBO1C372A was
generated by PCR-mediated mutagenesis (details available upon request).
The mutated PCR product was digested with EcoRI, and the
resulting EcoRI-EcoRI (nt 1105-1392 of HBO1)
fragment was cloned into the same sites of GAD-HBO1-(218-611). The DNA
sequence of all PCR-generated constructs was verified by sequencing.
pAdTrack-HA3MCM2 was constructed in multiple steps. The
mouse MCM2 cDNA was excised from pBS-mMCM2 (generous gift of H. Kimura, University of Oxford, Oxford, UK) as a
KpnI-NotI fragment and inserted into the
corresponding sites of pGEM-CMV (Nevins et al. (46)),
generating pREC-mMCM2. A NotI site was inserted immediately following the mMCM2 initiating methionine in a PCR using primers MCM2-NotI
(5'-CGGGGTACCATGAGCGGCCGCGCGGAGTCTTCTGAGTCT-3', initiator codon in bold and KpnI and NotI sites
underlined) and MCM2-427 (5'-CTGCCAGCCTCACGGTCCCG-3') which
amplify nts 51-427 of the mMCM2 cDNA. The PCR product was digested
with KpnI and BspEI and used to replace the
corresponding fragment in pREC-mMCM2 to generate
pREC-mMCM2-NotI. A fragment encoding three tandem copies of
the HA epitope was released from pMT252 (generous gift of M. Tyers,
Mount Sinai Hospital, Toronto, Canada) by digestion with
NotI and inserted into the 5' NotI site of
partially digested pREC-mMCM2-NotI to generate
pREC-HA3mMCM2. The entire PCR-amplified region of the
tagged mMCM2 was sequenced to confirm the construction and orientation.
pAdTrack-HA3MCM2 was constructed by excising the tagged
MCM2 construct from pREC-HA3MCM2 as an
Asp718-XbaI fragment and inserting it into the
corresponding sites of pAdTrack-CMV. pAdTrack-hORC1 was constructed by
excising human ORC1 from pcDNA3-hORC1 (44) as a
HindII-SmaI fragment and inserting into the
HindIII-EcoRV sites of pAdTrack-CMV.
AdTrack-AU5-HBO1 was constructed as follows. An N-terminal AU5 (TDFYLK)
epitope tag was added to the HBO1 cDNA by insertion of annealed
oligonucleotides TB43 (5'-
CTAGACCATGGCCACCGACTTCTACCTGAGGTT-3', initiating
codon in bold and XbaI site underlined) and TB44
(5'-TAAACTTCAGGTAGAAGTCGGTGGCCATGGT-3') into the XbaI and
NdeI sites of pBS-HBO1(rev), generating pBS-AU5-HBO1. An
Asp718-NotI fragment was excised from
pBS-AU5-HBO1 and ligated into the corresponding sites of pAdTrack-CMV,
creating AdTrack-AU5-HBO1.
Cells and Viruses--
C33A cervical carcinoma cells were grown
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and infected in 1.5 ml (per 100-mm plate) of Dulbecco's
modified Eagle's medium containing 25 mM HEPES (pH 7.5)
for 75 min at 37 °C. Following infection, Dulbecco's modified
Eagle's medium plus 10% fetal bovine serum was added, and cells were
incubated an additional 18 h prior to harvest. AdMCM2 (HA-tagged
mouse MCM2), AdORC1 (human ORC1), and AdHBO1 (AU5-tagged human HBO1)
recombinant adenoviruses were generated by the method of He et
al. (45) after subcloning into pAdTrack-CMV shuttle vector.
Adenoviral stocks were maintained as described (46) and purified by
cesium chloride density gradient centrifugation.
Yeast Two-hybrid Liquid Antibodies and Immunological Methods--
AdHBO1-produced HBO1
protein contains an N-terminal AU5 epitope tag and was detected using
purified monoclonal antibody
Immunoprecipitation experiments were performed by infecting human
cervical carcinoma C33A cells with recombinant adenoviruses as
indicated, and whole cell extracts were prepared by sonication (10 s)
in RIPA buffer (150 mM NaCl, 20 mM sodium
phosphate (pH 7.4), 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mM DTT, 0.1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin). Extracts were incubated with
antibodies GST Pull-down Experiments--
GST fusion protein expression
vectors (pGEX-dMCM2, -mMCM2, -dORC1, and -hORC1) were transformed into
E. coli strain BL21(DE3). Cells were grown to
A600 0.5-0.8, induced with 0.4 mM
isopropyl-1-thio- Chromatin-binding Assays--
C33A cells were harvested by
trypsinization and washed with phosphate-buffered saline. Cell pellets
were resuspended to a final volume of 1 ml of CSK+ buffer
(10 mM Pipes (pH 7), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.5%
Triton X-100, 1 mM ATP, 0.1 mM PMSF, 1 µg/ml
aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM
sodium orthovanadate, 25 mM Isolation of HBO1 as an MCM2-interacting Protein--
To gain
further insight into MCM function, we used a yeast two-hybrid system to
screen a human placental cDNA library for proteins that interact
with a Gal4 DNA-binding domain (GBD) fusion encoding the mouse MCM2
cDNA. Nine clones were identified by histidine nutritional
selection and HBO1 Interacts with MCM2 and ORC1 in Vitro and in Vivo--
To
confirm the HBO1 interaction with MCM2, we constructed full-length
HBO1-(1-611) as a Gal4 activation domain (GAD) fusion and compared its
binding activities to the C-terminal clone isolated in the
two-hybrid screen (HBO1-(218-611)). To generate a full-length HBO1
cDNA, a 0.7-kilobase N-terminal fragment of HBO1 was amplified from
a human placental cDNA library using gene-specific primers and
appended to clone TB305. As seen in Fig.
2A, the C-terminal clone of
HBO1-(218-611) specifically interacts with GBD fusions of mouse
(GBD-mMCM2) and Drosophila MCM2 (GBD-dMCM2), as well as
human (GBD-hORC1) and Drosophila ORC1 (GBD-dORC1), resulting in activation of the lacZ gene and production of
We sought to examine further the differential binding activities
observed in the yeast two-hybrid system and to determine whether
full-length HBO1 is capable of interacting with MCM2 and ORC1. To
accomplish this we performed GST pull-down experiments with in
vitro synthesized, [35S]methionine-labeled
full-length HBO1 protein and bacterially produced GST fusion proteins
GST-mMCM2, -dMCM2, -hORC1, and -dORC1. As seen in Fig.
2B, a strong and specific
retention of HBO1-(1-611) protein was observed for the samples
containing GST-mMCM2, -hORC1, and -dORC1, with a weaker retention
observed for GST-dMCM2. These findings demonstrate that full-length
HBO1 is capable of interacting with MCM2 and ORC1 in vitro
and indicate that the interactions of HBO1 with MCM2 and ORC1 are
evolutionarily conserved.
To confirm the interactions of HBO1 with MCM2 and ORC1 under
physiological conditions, we constructed recombinant adenoviruses expressing N-terminal AU5 epitope-tagged version of full-length human
HBO1 (AdHBO1), N-terminal HA-tagged full-length mouse MCM2 (AdMCM2),
and full-length human ORC1 (AdORC1). Human C33A cervical carcinoma
cells were infected with AdHBO1, AdMCM2, and AdORC1 (Fig. 3, as
indicated), and whole cell extracts were prepared and
immunoprecipitated with the indicated antibodies. The resulting immunoprecipitates were assayed for the presence of HBO1 or MCM2 by
immunoblot analysis with anti-AU5 (HBO1) or anti-HA (MCM2) antibodies,
respectively. In C33A cells, HBO1 polypeptide could be
coimmunoprecipitated with endogenous human MCM2 (Fig. 3A, left panel), demonstrating that the HBO1-MCM2 interaction is conserved with human MCM2 in living cells. This interaction was dependent upon
AdHBO1 infection of the cells and the presence of anti-MCM2 antibody in
the immunoprecipitation reaction. In a reciprocal experiment,
adenovirally produced mouse MCM2 protein coimmunoprecipitates with HBO1
(Fig. 3A, right panel). This interaction is dependent upon
the production of exogenous HA-tagged MCM2 and AU5-tagged HB01, as well
as anti-AU5 (HBO1) antibody. Finally, we confirmed the previously
reported interaction between human ORC1 and HBO1 (33) using ectopically
expressed ORC1 and HBO1 (Fig. 3B). Taken together, these
results confirm the existence of a conserved interaction between MCM2
and HBO1 in vivo and demonstrate that full-length HBO1
protein is capable of interacting with both MCM2 and ORC1.
HBO1 Localization--
Based on our findings that HBO1 interacts
with two components of the pre-RC in vitro and in
vivo, and the possibility that HBO1-associated HAT activity may be
important for the modulation of chromosomal histone function, we sought
to determine the cellular localization of HBO1 protein. To accomplish
this, we infected asynchronously growing C33A cells with AdHBO1 and
prepared nuclear and cytoplasmic extracts. As demonstrated previously
by immunofluorescent localization (48), immunoblot analysis revealed
that ectopically expressed HBO1 protein is localized exclusively to the
nuclear fraction (Fig. 4A, upper
panel). By comparison, endogenous MCM2 protein is found both in
the nuclear and cytoplasmic fractions (Fig. 4A, middle
panel). Furthermore, we found that a fraction of the exogenous
HBO1 protein is associated with a chromatin-enriched, low speed pellet
(Fig. 4B), and that chromatin-associated HBO1 is resistant
to release by nuclease digestion (data not shown). The nuclease
resistance of chromatin-associated HBO1 protein is reminiscent of a
study demonstrating that chromatin-bound human ORC is resistant to
release by nuclease digestion (49). The observation that HBO1 protein
is nuclear and associated with a chromosomal fraction is consistent
with a role in regulation of chromatin dynamics, although the
significance of the nuclease resistance of HBO1 and ORC remains to be
determined.
HBO1 Zinc Finger Is Required for Binding to MCM2 and ORC1--
We
have demonstrated that HBO1 interacts with MCM2 both in
vitro and in vivo. However, the possibility remains
that the interaction between MCM2 and HBO1 is mediated by ORC1 or that
the ORC1-HBO1 interaction is mediated by MCM2. Therefore, we mapped the
interaction domains of HBO1 and MCM2 to characterize further these
interactions. The highly conserved MYST domain of HBO1 is defined by
the presence of two distinct structural motifs, an atypical
C2HC zinc finger and an acetyl-CoA-binding motif. Each
member of the MYST family (except Esa1p) (40) conforms to the
Cys-X2-Cys-X12-His-X3-Cys consensus. Single zinc fingers of functional significance are less
common than repeated zinc finger motifs (50). Likewise, C2HC motifs are less common than other zinc finger motifs
and typically occur singly (51), or as the first or last of a series of
zinc fingers (52, 53). The significance of this unusual motif remains
to be determined, although zinc fingers may function in nucleic acid
binding, and single zinc fingers are most likely to facilitate
oligomerization or protein-protein interactions (50).
To map the region of HBO1 responsible for binding to MCM2 and ORC1, and
to determine whether the C2HC zinc finger plays a role in
these interactions, a series of HBO1 deletion mutants fused to the GAD
were constructed, cotransformed into yeast strain Y190 with plasmids
encoding GBD-dMCM2, -mMCM2, or -hORC1, and assayed for
To determine whether the C2HC zinc finger motif plays a
role in protein-protein interactions with MCM2 and ORC1, we mutated the
second cysteine of the zinc finger to an alanine (Fig. 5, GAD-HBO1C372A). This single amino acid substitution
abolished the ability of HBO1 to interact with dMCM2, mMCM2, and hORC1. These findings indicate that the C2HC zinc finger is
necessary, but not sufficient, for interactions with MCM2 and ORC1 and
provide insights into the functional role played by this highly
conserved structural motif. Furthermore, these studies highlight the
conserved nature of the protein-protein interactions of HBO1 with
Drosophila and mouse MCM2, as well as with human ORC1.
An N-terminal Domain of MCM2 Is Required for Binding to
HBO1--
To map the region of MCM2 responsible for binding to HBO1, a
series of MCM2 deletion mutants fused to the GBD were constructed and
assayed for HBO1 binding activity (Fig.
6). Given the highly conserved mechanism
of interaction between HBO1 and MCM2, and the potential for translating
observations made in these studies into a system that would allow
future genetic analyses, Drosophila MCM2 was used for these
analyses. In this two-hybrid assay, the first 72 amino acids of dMCM2
(GBD-dMCM2-(73-888)) were found to be dispensable for binding to HBO1.
Further N-terminal truncations (GBD-dMCM2-(106-888) and
GBD-dMCM2-(133-888)) resulted in an incremental decrease in HBO1
binding to ~10% of full-length dMCM2. Finally, fusion proteins
lacking the first 223 amino acids of dMCM2 were completely deficient in
Similarly, removal of C-terminal residues through amino acid 224 (GBD-dMCM2-(1-861), GBD-dMCM2-(1-668), GBD-dMCM2-(1-467), GBD-dMCM2-(1-234), and GBD-dMCM2-(1-223)) had relatively small effects on Isolation of an Allele of MCM2 Deficient in HBO1
Interaction--
Having mapped the region of MCM2 important for
interacting with HBO1 to the N terminus of MCM2 (amino acids 72-223),
we wished to generate a mutant MCM2 allele defective for the ability to interact with HBO1. To accomplish this, we employed a reverse yeast
two-hybrid system (42, 56). This system employs an engineered yeast
strain in which the interaction of two proteins expressed in the
context of a two-hybrid system is deleterious to growth. Under these
conditions, the mutation of a residue that is critical for this
interaction provides a selective growth advantage, thereby facilitating
detection. For this we used the counter-selectable yeast gene
URA3, which encodes an enzyme involved in uracil
biosynthesis, and also catalyzes the conversion of the nontoxic analog
5-FOA to a toxic product 5-fluorouracil. The expression level of this URA3 marker is tightly coupled to the formation of a
functional transcription factor (through a pair of interacting GAD/GBD
fusion proteins) and thus confers a Ura+ 5-FOA-sensitive
(5-FOAS) phenotype in the presence of transcriptional
activity and Ura
To generate a large library of random mutations in the region of dMCM2
previously identified as critical for HBO1 interaction, we performed
PCR amplification of the cDNA encoding this region under conditions
that favor nucleotide misincorporation (mutagenic PCR) (42). The
mutagenized PCR fragment was then cotransformed into yeast cells with
linearized GBD-dMCM2 expression vector containing homologous flanking
sequences but lacking the HBO1-interacting region to allow
incorporation by homologous recombination. Mutant allele
MCM2R2H101 was isolated from this mutagenized
GBD-dMCM2 library in the presence of GAD-HBO1-(218-611) and was found
to contain three point mutations resulting in amino acid substitutions (R114S, V171A, and L222P). As shown in Fig.
7A, clone
GBD-dMCM2R2H101 is unable to interact with HBO1, resulting
in a 5-FOAR phenotype and no
Alignment of MCM2 amino acid sequences from nine eukaryotic species as
well as the archaeal MCM protein reveals that leucine residues occupy
the equivalent position in each species (Fig. 7B),
suggesting a functional importance for this leucine. To test whether this same leucine to proline mutation has a similar effect on
HBO1 binding in the context of the mouse MCM2 protein, we generated a
GBD-mMCM2L237P mutant, and we assayed its ability to
interact with HBO1 in the yeast two-hybrid system (Fig. 7C).
As seen with the Drosophila MCM2 protein, the leucine to
proline mutation at this position of mouse MCM2 results in a 13-fold
decrease in
The leucine at position 222 of Drosophila MCM2 is predicted
to be located in a turn region immediately flanked by an upstream Isolation of an HBO1 Mutation That Restores Interaction with the
dMCM2L222P Mutant--
We wished to obtain definitive
evidence confirming that the interaction between MCM2 and HBO1 is
direct and not mediated by a "bridging" protein such as ORC1. A
classical genetic method of demonstrating a direct interaction between
two proteins is to isolate a mutant allele of one protein that disrupts
the interaction, and then to isolate a second site mutation in its
partner protein that restores the ability to interact with the
interaction-defective allele. We have demonstrated that mutant
dMCM2L222P is defective for the ability to interact with
HBO1. By using a library of random mutations spanning the entire HBO1
coding sequence of GAD-HBO1-(218-611), we screened for a mutant that
restores the interaction with dMCM2L222P in a yeast
two-hybrid system. From this screen, a single clone (GAD-HBO1SSS1) was isolated and found to contain three
mutations resulting in amino acid changes (K282R, I380T, and Y567H). As shown in Fig. 8, GAD-HBO1SSS1
was capable of partially restoring dMCM2 interaction, as measured by
The MCM family of proteins are conserved in all eukaryotes and are
essential for normal DNA replication. Nevertheless, the precise
function of MCMs in DNA replication remains to be fully understood. In
this study, we identified a novel interaction between the MCM2 protein,
and HBO1, a putative histone acetyltransferase and MYST domain protein.
These data, together with the previously characterized interaction
between ORC1 and HBO1 (33), suggest that human HBO1 protein may play a
direct role in DNA replication.
Several lines of evidence support the notion that the interaction
between MCM2 and HBO1 is specific, physiologically significant, and
evolutionarily conserved. First, the MCM2-HBO1 interaction was detected
in several assay systems, including yeast two-hybrid, GST pull-down,
and coimmunoprecipitation in human cells. Second, we have generated
interaction-defective alleles of dMCM2 (dMCM2L222P) and
HBO1 (HBO1C372A) that are defective in their ability to
bind to their wild-type partner protein, respectively. Additionally, we
have demonstrated a direct interaction between MCM2 and HBO1 by
generating a mutant version of HBO1 (HBO1I380T) that
restores the ability to interact with dMCM2L222P. This
second site suppressor mutation serves as genetic evidence of a direct
interaction between MCM2 and HBO1.
The previous finding that HBO1 interacts with ORC1, together with the
data presented here demonstrating a direct interaction with MCM2,
suggests the possibility that HBO1 might be recruited to a
pre-replication complex by multiple protein interactions. For the
reasons stated above, we believe that these are likely independent
interactions involving MCM2 and ORC1. Nevertheless, it is also quite
possible that they are synergistic, resulting in the formation of a
more stable complex involving HBO1.
Histone acetylation serves as a general mechanism to destabilize
nucleosome core particles during various processes occurring in
chromatin. Allfrey et al. (57) first reported that addition of acetyl groups to histone proteins is associated with
transcriptionally active regions of the genome. More recently, many
proteins that regulate transcription have been shown to possess or
recruit HAT or histone deacetylase activities (reviewed in Ref. 58).
Several MYST subfamily proteins have been implicated in a variety of
transcriptional processes including transcriptional activation, dosage
compensation, and silencing. MOZ was discovered due to its involvement
in a chromosomal translocation that fuses MOZ to the transcriptional coactivator CREB-binding protein, resulting in a form of acute myeloid leukemia (34). Tip60 was cloned by its ability to interact with
the viral activator HIV Tat in a two-hybrid system (36). MOF was
discovered in a genetic screen for gene products necessary for
hypertranscription and the acetylation of histone H4 at Lys-16 that is
associated with Drosophila dosage compensation (37). Interestingly, the mof mutant identified in this screen
contains a single point mutation located in the acetyl-CoA-binding
(HAT) motif. SAS2 and SAS3 were identified as enhancers of
sir1 epigenetic silencing defects in S. cerevisiae (38), and Esa1 was subsequently isolated by homology to
SAS2 and SAS3 and shown to modulate genes important for cell cycle
control (40, 59). Among these proteins, Tip60 and Esa1 have been shown
to possess intrinsic HAT activity (40, 60).
The result of histone acetylation is a change in chromatin structure
and a corresponding increase in the accessibility of the DNA by
trans-acting factors. Given this notion, it might be expected that
other DNA-dependent enzymatic activities requiring access
to chromosomal DNA would also benefit from chromatin modification by
histone acetylation, and this appears to be the case. McMurry and
Krangel (61) recently used an antibody to acetylated histone H3 in a
chromatin immunoprecipitation assay to demonstrate a link between
histone acetylation and V(D)J recombination in the TCR By analogy, destabilization of chromatin structure through histone
acetylation might also play an important role in the regulation of DNA
replication. In proliferating rat liver cells, an increase in HAT
activity is associated with the onset of DNA replication (63), and
depression of histone acetylation by alkylating agents correlates with
an inhibition of thymidine incorporation (64). Additionally, studies
using Chinese hamster chromosomes demonstrate a close correlation
between low levels of histone H4 acetylation and late DNA replication
(65). More directly, core histone acetylation has been demonstrated
previously to play a role in DNA replication efficiency (66). In this
study, an SV40 in vitro replication system was used to
analyze the replication efficiencies of SV40 minichromosomes associated
with normal or hyperacetylated histones. Elongation of replication
occurred with a higher efficiency in hyperacetylated minichromosomes,
indicating that movement of the replication machinery through
nucleosomal DNA is facilitated by acetylation of histone tails. This
potential role of histone acetylation in the elongation stage of DNA
replication is consistent with the finding that MCM proteins migrate
with the replication fork during elongation (67). Additionally, Aoki
and Schultz (68) recently examined the temporal and spatial
distribution of sites of DNA replication in one-cell mouse embryos, and
they demonstrated that inhibition of transcription with Although the results presented here and elsewhere provide evidence for
protein-protein interactions that could recruit a histone acetylase to
a pre-replication complex, this possibility remains only a hypothesis
that must be tested in the context of relevant biological systems. We
believe the best approach to this question is a genetic strategy that
might correlate potential physical interaction with biological
function. Toward this end, our isolation of alleles of
Drosophila and mouse MCM2 (dMCM2L222P and
mMCM2L237P, respectively) that are defective for
interaction with HBO1 provides an approach to this question. This
interaction-defective allele can then be compared with its wild-type
counterpart for the ability to functionally complement a knock-out in
the corresponding gene or for the ability to function in an expression
assay in the relevant cells. By taking this functional analysis one
step further, it might then be possible to isolate a second site
suppressor mutation, such as HBO1I380T, that restores the
ability of the interaction-defective allele to interact. This second
site suppressor might then be used to functionally rescue the
interaction-defective allele, restoring the wild-type phenotype.
Clearly, a physiological link between HAT activity and origin function
is an intriguing possibility, and establishing that link by functional
assays is a critical next step in this investigation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity. Approximately 200 His+ clones were tested for growth on synthetic complete
plates lacking tryptophan, leucine, and histidine (Sc-T-L-H) containing
5 mM 3-amino-1,2,4-triazole to select for strong
interactions. Plasmid DNA from nine clones were isolated from yeast,
transformed into the Escherichia coli strain MH1066, and
plated on M9 (leu
) plates. Leu+ plasmid DNA
was isolated and re-transformed into HF7c harboring GBD-mMCM2 to
confirm the interaction with MCM2. cDNA clones encoding putative
mMCM2-interacting proteins were sequenced and compared against the
National Center for Biotechnology Information data bases using the
basic local alignment search tool (BLAST) algorithm.
,
gal80
, SPAL10::URA3, GAL1::LacZ,
GAL1::HIS3@LYS2, can1R,
cyh2R; Vidal et al. (56)) carrying
GAD-HBO1-(218-611), and the PCR products were integrated
in vivo by homologous recombination (41, 42). Approximately
100,000 Leu+ Trp+ transformants were plated on
15 cm Sc-T-L plates containing 0.1% 5-fluoroorotic acid (5-FOA) where
~200 5-FOA-resistant (5-FOAR) colonies developed.
Trp+ plasmid DNA from 20 5-FOAR clones was
rescued and re-transformed into MAV103 containing GAD-HBO1-(218-611)
and assayed to confirm 5-FOA resistance. Yeast extracts were prepared
from 5-ml Sc-T-L cultures and assayed for the production of full-length
GBD-dMCM2 fusion protein by Western blot using a monoclonal antibody
against Gal4 (DBD) (RK5C1; Santa Cruz Biotechnology). Four
interaction-defective alleles of dMCM2 producing full-length fusion
proteins were sequenced to identify point mutations. Single point
mutations were generated using the GeneEditor site-directed mutagenesis
kit (Promega, Madison, WI).
, gal80
, cyhr2,
LYS2:: GAL1UAS-HIS3TATA-HIS3,
URA3::GAL1UAS-GAL1TATA-lacZ) harboring plasmid GBD-dMCM2L222P. Approximately 6000 Leu+ Trp+ transformants were plated on 15-cm
Sc-T-L-H plates containing 15 mM 3-amino-1,2,4-triazole.
Developing colonies were screened for
-galactosidase activity, and a
single colony was positive. Leu+ plasmid DNA was rescued
and re-transformed into Y190 containing GBD-dMCM2L222P, and
the interaction was confirmed by liquid
-galactosidase assay and
sequenced to identify point mutations. Single point mutations were
generated using the GeneEditor site-directed mutagenesis kit
(Promega).
-Galactosidase
Assays--
Quantitative two-hybrid liquid culture
-galactosidase
assays (47) were performed in yeast strains Y190 or MAV103. Expressions levels of the GAD and GBD fusion proteins were controlled by Western blot analysis for each experiment. Experiments were performed a minimum
of three times. Values shown are representative results from individual experiments.
-AU5 (Babco, Richmond, CA). Native MCM2
was detected with mouse polyclonal antibody
-BM28 (Transduction
Laboratories, Lexington, KY) against human MCM2. AdMCM2-produced mouse
MCM2 protein containing an N-terminal influenza hemagglutinin (HA)
epitope tag was detected using polyclonal antibody
-HA (Y-11; Santa
Cruz Biotechnology, Santa Cruz, CA) or
-BM28, as indicated.
Endogenous and AdORC1-produced human ORC1 was detected using monoclonal
antibody
-ORC1 (7F6/1, Novus Biologicals, Littleton, CO) against
human ORC1. GBD and GAD fusion proteins were detected using monoclonal
antibody
-Gal4 (GBD) (RK5C1; Santa Cruz Biotechnology) and
-HA
(Y-11, Santa Cruz Biotechnology), respectively.
-BM28,
-HA,
-ORC1, or
-AU5 for 1 h at
4 °C. Protein G Plus/protein A-Agarose (Calbiochem) was added and
incubation continued for 1 h at 4 °C. After extensive washes
with RIPA buffer, immunoprecipitated proteins were resolved by
SDS-polyacrylamide gel electrophoresis and detected by immunoblotting and fluorography.
-D-galactopyranoside at 30 °C for
1-2 h, and collected by centrifugation. Cell pellets were resuspended
in buffer (25 mM Tris (pH 7.9), 50 mM NaCl, 0.1 mM EDTA, 5% glycerol, 1 mM DTT, 0.1 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin), sonicated, and Triton X-100 added to 1% final.
Following a 30-min incubation on ice, insoluble material was removed by
centrifugation (5 min at 3,000 × g). Soluble extracts
were incubated with glutathione-Sepharose 4B (AP Biotech) for 30 min at
room temperature, rinsed extensively with CoIP buffer (50 mM HEPES (pH 7.2), 125 mM potassium acetate,
0.5 mM EDTA (pH 8), 0.5 mM EGTA (pH 8), 0.1%
Tween 20, 12.5% glycerol, 1 mM DTT, 0.1 mM
PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin), and resuspended in CoIP buffer. Full-length HBO1 protein was
transcribed and translated in vitro using the TNT
reticulocyte lysate system (Promega) with
[35S]methionine, T3 polymerase, and plasmid
pBS-HBO1-(1-611). The products were precipitated with an equal volume
of saturated ammonium sulfate (pH 7.4) at 4 °C for 10 min and
collected by centrifugation (5 min at 10,000 × g).
Insoluble material was washed once with 2 M ammonium
sulfate (pH 7.4) and resuspended in CoIP buffer. Equal amounts of GST,
GST-dMCM2, -mMCM2, -dORC1, and -hORC1 coupled to glutathione-Sepharose
beads were incubated with 35S-HBO1 protein for 30 min at
room temperature in CoIP buffer. Beads were gently washed five times in
CoIP buffer, boiled in SDS sample buffer, and analyzed by 10% SDS-PAGE
followed by autoradiography.
-glycerol phosphate). Fifty
microliters of whole cell lysate (5%) was reserved for total protein
control. Lysates were incubated on ice for 20 min, and then centrifuged
at low speed for (1,000 × g) for 5 min at 4 °C.
Chromatin pellets were washed once with 1 ml of CSK+ buffer
for 5 min on ice and centrifuged again (1,000 × g).
Chromatin-containing pellets were resuspended in SDS sample buffer,
resolved by SDS-PAGE, transferred to nitrocellulose membrane, and
analyzed by immunoblotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity. Sequence analysis revealed
that two of these clones (TB305 and TB310) were identical to the C
terminus (aa 218-611) of a protein recently identified as interacting
with ORC1, termed HBO1 (33). HBO1 was subsequently shown to interact
with human androgen receptor and modulate androgen receptor
transcriptional activity in a ligand-dependent manner (48).
HBO1 is a 611-amino acid protein with a predicted molecular mass of 70 kDa (estimated pI 9.2) and contains a unique N-terminal serine-rich
region (22% in aa 1-167) and a conserved 270-amino acid C-terminal
domain characteristic of the MYST family of proteins (Fig.
1A) (34). The MYST domain
contains a putative acetyl-coenzyme A (acetyl-CoA)-binding domain (Fig.
1B) and an atypical C2HC zinc finger. Throughout
the MYST domain, HBO1 is highly conserved (~40-65% identical
and 60-80% similar) to several other MYST family proteins (Fig.
1A).
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Fig. 1.
The MYST family of histone
acetyltransferases. A, schematic representation of putative
MYST family acetyltransferase proteins human (Hs) HBO1, MOZ,
MORF, and Tip60, Drosophila (Dm) MOF, HAT
(GenBankTM accession number AAF44628), EG0007.7
(GenBankTM accession number AAF45923), S. cerevisiae (Sc) Esa1, SAS2, and SAS3, and S. pombe (Sp) HAT (GenBankTM accession number
CAA22591). Zinc finger (C2HC and C4HC), histone
acetyltransferase (HAT), transcriptional repression
(TR), and activation (TA), acidic, serine
(S)- and glutamine (Q)-rich, and chromodomain
(chromo) motifs are indicated. Percent conservation within
the MYST domain relative to HsHBO1 is indicated for each
protein (similarity/identity). B, amino acid alignment of
the acetyl coenzyme A-binding motif for MYST family members.
-galactosidase activity. Interestingly, whereas the full-length
GAD-HBO1 interacts relatively well with GBD-hORC1, it does not appear
to produce
-galactosidase activity when coexpressed with the
GAD-mMCM2, -dMCM2, and -dORC1 fusion proteins. Although this may be due
to an inability of the full-length GAD-HBO1-(1-611) fusion protein to
bind to mMCM2, dMCM2, and dORC1 in this system, it may also reflect the
presence of an intrinsic transcriptional repression domain located in
the N-terminal region of HBO1 (48). This N-terminal transcriptional
repression activity may prevent detectable interactions when monitored
in a transcription-based assay such as the yeast two-hybrid system and
may become masked or inactivated when coexpressed with
GBD-hORC1.
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Fig. 2.
HBO1 interacts with MCM2 and ORC1 in
vitro. A, analysis of partial versus
full-length HBO1 protein-protein interactions with MCM2 and ORC1.
Partial (aa 218-611) and full-length (aa 1-611) HBO1 fused to the
GAL4 activation domain (GAD) were individually cotransformed
into yeast strain Y190 with constructs encoding the GAL4 DNA-binding
domain alone (GBD) or GBD fused to full-length mouse MCM2
(mMCM2), Drosophila MCM2 (dMCM2),
human ORC1 (hORC1), or Drosophila ORC1
(dORC1), as indicated. Transformants were grown in selective
medium, and -galactosidase (
-gal) activity
was measured in a quantitative liquid culture assay. B, HBO1
was synthesized in vitro (35S-labeled) and
incubated with GST or GST-mMCM2, -dMCM2, -hORC1, or -dORC1 fusion
proteins coupled to Sepharose beads and washed extensively. Proteins
were resolved on 10% SDS-PAGE gel and analyzed by autoradiography.
10% of input HBO1 protein is indicated in left panel.
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Fig. 3.
HBO1 binds to MCM2 and ORC1 in
vivo. A, asynchronously growing C33A cervical
carcinoma cells were infected with either AdMCM2 (multiplicity of
infection (m.o.i.), 25 focus-forming units/cell), AdHBO1 (m.o.i.
50), AdORC1 (m.o.i. 300), or uninfected, as indicated. Following
immunoprecipitation (IP) from C33A whole cell extract with
either -MCM2 (left) or
-AU5 antibodies
(right), as indicated, coprecipitated proteins were resolved
by SDS-PAGE, transferred to nitrocellulose membrane, and probed with
-AU5 (left) or
-HA (right) antibodies. C33A
extract (5% of input) is shown for AdHBO1 (left) and AdMCM2
(right) infection. B, following
immunoprecipitation from C33A extract with
-ORC1 antibodies,
coprecipitated proteins were resolved by SDS-PAGE, transferred to
nitrocellulose membrane, and probed with
-AU5 antibodies.
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Fig. 4.
HBO1 is nuclear- and chromatin-associated.
A, asynchronously growing C33A cells were infected with
AdHBO1 (m.o.i. 50) or uninfected, as indicated. Nuclear and cytoplasmic
fractions were prepared as described previously (69) and resolved by
SDS-PAGE, transferred to nitrocellulose membrane, and probed with
-AU5 (upper panel),
-MCM2 (middle panel),
or
-actin (lower panel) antibodies. B,
asynchronously growing C33A cells were infected with AdHBO1 (m.o.i. 10 or 50) or uninfected, as indicated. Cells were harvested 18 h
post-infection by trypsinization and lysed with 0.5% Triton X-100.
Five percent of the total protein was reserved, and the remainder was
subjected to low speed centrifugation to produce a chromatin-enriched
pellet fraction. Chromatin-bound (upper panel) and total
protein (lower panel) fractions were resolved by SDS-PAGE,
transferred to nitrocellulose membrane, and probed for HBO1 using
-AU5 antibody.
-galactosidase activity (Fig. 5). As
observed previously in Fig. 2A, GBD-dMCM2, -mMCM2, and
-hORC1 interacted strongly with GAD-HBO1-(218-611), whereas only hORC1
produced activity in conjunction with full-length GAD-HBO1-(1-611).
Interestingly, the relative ability of each of the remaining GAD-HBO1
deletion mutants to interact with GBD-dMCM2, -mMCM2, and -hORC1 varied in a parallel manner. Removal of the C-terminal 69 amino acids of HBO1
(GAD-HBO1-(218-542)) significantly reduced
-galactosidase activity
for each GBD fusion protein. Further truncation of the C terminus
through amino acids 399 (GAD-HBO1-(218-470) and GAD-HBO1-(218-398)) resulted in a further decrease in activity (1-5% relative to GAD- HBO1-(218-611)). Finally, removal of an additional 31 amino acids, including the C2HC putative zinc finger
(GAD-HBO1-(218-367)), completely abolished the ability of HBO1 to
interact with dMCM2, mMCM2, and hORC1 (as measured by
-galactosidase
activity). In this two-hybrid assay, amino acids 1-259 were found to
be dispensable for binding to dMCM2, mMCM2, and hORC1. Further
N-terminal truncations (GAD-HBO1-(320-611)) completely abolished the
ability to interact with each of the GBD fusion proteins. Thus, a
region encompassing much of the highly conserved MYST domain, including
the C2HC zinc finger, is required for binding to MCM2 as
well as ORC1.
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Fig. 5.
Mapping the region of HBO1 that
interacts with MCM2 and ORC1. Human HBO1 deletion mutants fused to
the GAD were individually cotransformed into yeast strain Y190 with
constructs encoding full-length GBD-dMCM2, -mMCM2, or -hORC1.
Transformants were grown in selective medium, and -galactosidase
activity was measured in a quantitative liquid culture assay.
-Galactosidase (
-gal) activity of each
GAD-HBO1 deletion construct is shown as a percentage relative to the
activity of the respective full-length GBD fusion protein with
GAD-HBO1-(218-618). Schematic representations of each GAD-HBO1
deletion construct are shown. Numbers correspond to amino
acid residues. Highly conserved MYST domain, C2HC zinc
finger, and acetyl coenzyme A-binding motif (HAT) are
indicated.
-galactosidase activity, indicating that this N-terminal region is
critical for MCM2-HBO1 interaction.
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Fig. 6.
Mapping the region of MCM2 that interacts
with HBO1. Drosophila MCM2 deletion mutants fused to
the GBD were individually cotransformed into yeast strain Y190 with
construct GAD-HBO1-(218-611). Transformants were grown in selective
medium, and -galactosidase (
-gal) activity
was measured in a quantitative liquid culture assay.
-Galactosidase
activity of each deletion construct is graphed as a percentage relative
to the activity of full-length GBD-dMCM2 with GAD-HBO1-(218-618).
Schematic representations of each GBD-dMCM2 deletion construct are
shown. Numbers correspond to amino acid residues. Known structural and
functional motifs, including histone-binding domain (54), zinc fingers,
and ATP-binding (ATPase) motifs (55), are indicated.
-galactosidase activity, indicating that amino acids 224-888 are dispensable for interactions with HBO1. C-terminal truncation through amino acid 106 (GBD-dMCM2-(1-105)) further reduced
HBO1 binding activity to less than 50% of full-length dMCM2.
Consistent with the absence of an effect upon removal of the N-terminal
72 amino acids of dMCM2 (GBD-dMCM2-(72-888)) discussed previously,
amino acids 1-72 (GBD-dMCM2-(1-72)) were devoid of HBO1 binding
activity. Finally, an internal fragment of dMCM2 encoding amino acids
72-223 (GBD-dMCM2-(72-223)) was capable of binding HBO1 resulting in
27% of the level of full-length dMCM2
-galactosidase activity. This
finding demonstrates that amino acids 72-223 are necessary and
sufficient for interacting with HBO1. We also found that a similar
fragment of mouse MCM2 (GBD-mMCM2-(1-257)) retains full HBO1 binding
activity relative to full-length mMCM2 (data not shown). This
HBO1-interacting region of MCM2 overlaps the previously mapped histone
H3-binding domain of human MCM2 (54) (Fig. 5) and is upstream of the
putative zinc finger motifs and the highly conserved central MCM core
domain encompassing an element similar to the Walker-type
NTP-binding sequence (55).
5-FOA-resistant (5-FOAR)
phenotype in its absence. Thus, in a large population of cells that
express wild-type interacting hybrid proteins (5-FOAS
phenotype), a few cells in which this interaction is disrupted can be
selected on the basis of their 5-FOAR phenotype.
-galactosidase activity
(compare rows 2 and 3). By introducing each of
the three mutations individually into GBD-dMCM2, we determined that the
leucine to proline mutation at position 222 of dMCM2 is responsible for
the observed interaction-defective phenotype (Fig. 7A,
compare rows 4-6).
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Fig. 7.
Identification and characterization of an
interaction-defective allele of MCM2. A, yeast strain MAV103
harboring the construct GAD-HBO1-(218-611) was individually
transformed with GBD, GBD-dMCM2, or mutants
GBD-dMCM2R2H101, -dMCM2R114S,
-dMCM2V171A, or -dMCM2L222P, as indicated.
5-Fold serial dilutions of each transformant were plated on selective
media without or with 0.05% 5-FOA. Additionally, transformants were
grown in selective medium, and -galactosidase
(
-gal) activity was measured in a quantitative
liquid culture assay. B, the leucine residue at amino acid
position 222 of Drosophila MCM2 is conserved throughout
eukaryotes (bold). Identical (dark) and similar
(light) residues are boxed and shaded.
Aligned protein sequences include the corresponding regions from
Drosophila (Dm) MCM2-(217-227), mouse
(Mm) MCM2-(232-242), human (Hs) MCM2-(224-234),
Xenopus (Xl) MCM2-(217-227),
Caenorhabditis elegans (Ce) MCM2-(206-216),
S. cerevisiae (Sc) MCM2-(244-254), S. pombe (Sp) MCM2-(237-247), Aspergillus
nidulans (An) MCM2-(241-251), Entamoeba
histolytic (Eh) MCM2-(293-303), and
Methanobacterium thermoautotrophicum (Mt)
MCM-(42-52). C, yeast strain Y190 containing
GAD-HBO1-(218-611) was transformed with either GBD-mMCM2 or
GBD-mMCM2L237P, grown in selective medium, and
-galactosidase activity was measured in a quantitative liquid
culture assay. D, yeast strain Y190 containing
GAD-HBO1-(218-611) was transformed with either GBD-dMCM2,
GBD-dMCM2L222P, or GBD-dMCM2L222A, grown in
selective medium, and
-galactosidase activity measured in a
quantitative liquid culture assay.
-galactosidase activity.
-sheet and a downstream
-helix (Chou-Fasman secondary structure analysis; MacVector 6.5.3). Insertion of a proline residue (which has a
low degree of conformational freedom since the proline side chain
nitrogen atom is covalently bonded to the main chain forming a rigid
ring) at this position might be expected to produce a rigid
conformational change in the secondary structure of this critical
region of MCM2. As such, we constructed a more subtle leucine to
alanine point mutation at position 222 of Drosophila MCM2
(GBD-dMCM2L222A), and we assayed its ability to interact with HBO1 in the two-hybrid system. As shown in Fig. 7D,
GBD-dMCM2L222A, like GBD-dMCM2L222P, is
defective for interaction with HBO1-(218-611), suggesting that the
absence of leucine at position 222, and not the insertion of a proline,
is the critical determinant for disrupting HBO1 binding.
-galactosidase activity. Construction and analysis of each of the
GAD-HB01SSS1 point mutations individually indicates that a
single amino acid change is responsible for the observed phenotype.
Mutant GAD-HBO1I380T restored the ability to interact with
GBD-dMCM2L222P to ~50% of the level of the wild-type MCM2 and HBO1 proteins. Interestingly, this isoleucine to threonine suppressor mutation maps to the X12 spacer
region of the C2HC zinc finger of HBO1. This is consistent
with the finding that disruption of the second cysteine residue of the
zinc finger (HBO1C372A, Fig. 5) abolishes the ability of
HBO1 to interact with MCM2 and ORC1 and further strengthens the notion
that the C2HC zinc finger of HBO1 plays a critical role in
direct protein-protein interactions with MCM2.
View larger version (21K):
[in a new window]
Fig. 8.
HBO1 second site suppressor mutation.
Yeast strain Y190 was cotransformed with wild-type GBD-dMCM2 and
GAD-HBO1-(218-611) or with mutant GBD-dMCM2L222P and
either wild-type GAD-HBO1-(218-611) or mutants
GAD-HBO1SSS1, -HBO1K282R,
-HBO1I380T, or -HBO1Y567H, as indicated.
Transformants were grown in selective medium, and -galactosidase
(
-gal) activity was measured in a quantitative
liquid culture assay.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
locus.
Furthermore, targeted deletion of the TCR
enhancer eliminates TCR
locus hyperacetylation and completely blocks V-to-J
rearrangement. Whereas the correlation between histone acetylation and accessibility to the V(D)J recombinase is striking, it remains to be determined how
histone acetylation alters accessibility to specific gene loci, and
whether altered enzymatic activity is a cause or effect of differential
accessibility. Histone acetylation also appears to play an important
role in DNA repair. Ectopic expression of mutated MYST domain protein
Tip60 lacking histone acetylase activity results in cells with
defective double-strand DNA break repair and impaired apoptotic
competence, indicating that Tip60-associated HAT activity plays a role
in DNA repair and apoptosis (62).
-amanitin
had no effect on the patterns of DNA replication. However, treatment of
embryos with a specific inhibitor of histone deacetylase accelerated
the completion of replication. This study suggests that acetylation of
histones, but not transcriptional activity, is involved in the
regulation of DNA replication in one-cell mouse embryos. These findings, along with the observations that HBO1 interacts with two
components of the replication machinery, MCM2 and ORC1, make it
interesting to speculate that HBO1-associated HAT activity may play a
direct role in modulation of DNA replication.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Carol Newlon for helpful advice and discussions throughout this work, to Amber Engel for technical support, and to Kaye Culler for assistance in the preparation of the manuscript.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by an American Cancer Society Fellowship
PF-99-111-01-CCG.
§ Supported by an American Cancer Society Fellowship PF-4465.
¶ Supported by the Viral Oncology Training Grant CA09111-25 from the National Institutes of Health.
Investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed: Tel.: 919-684-2746; Fax: 919-681-8973; E-mail: j.nevins@duke.edu.
Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M011556200
2 T. W. Burke, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: ORC, origin recognition complex; MCM, minichromosome maintenance; HBO1, histone acetyltransferase bound to ORC1; pre-RC, pre-replicative complex; HAT, histone acetyltransferase; MOZ, monocytic leukemia zinc finger protein; MORF, MOZ-related factor; Tip60, HIV Tat-interacting protein; HIV, human immunodeficiency virus; MOF, males absent on the first; SAS, something about silencing; Esa1, essential SAS-related acetyltransferase; GST, glutathione S-transferase; GAD, Gal4 activation domain; GBD, Gal4 DNA-binding domain; m.o.i., multiplicity of infection; 5-FOA, 5-fluoroorotic acid; HA, hemagglutinin; PCR, polymerase chain reaction; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; aa, amino acids; Pipes, 1,4-piperazinediethanesulfonic acid; nt, nucleotide.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Dutta, A., and Bell, S. P. (1997) Annu. Rev. Cell Dev. Biol. 13, 293-332[CrossRef][Medline] [Order article via Infotrieve] |
2. | Newlon, C. S. (1997) Cell 91, 717-720[CrossRef][Medline] [Order article via Infotrieve] |
3. | Donaldson, A. D., and Blow, J. J. (1999) Curr. Opin. Genet. & Dev. 9, 62-68[CrossRef][Medline] [Order article via Infotrieve] |
4. | Bell, S. P., and Stillman, B. (1992) Nature 357, 128-134[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Stillman, B.
(1994)
J. Biol. Chem.
269,
7047-7050 |
6. | Cocker, J. H., Piatti, S., Santocanale, C., Nasmyth, K., and Diffley, J. F. X. (1996) Nature 379, 180-182[CrossRef][Medline] [Order article via Infotrieve] |
7. | Diffley, J. F. X. (1996) Genes Dev. 10, 2819-2830[CrossRef][Medline] [Order article via Infotrieve] |
8. | Dahmann, C., Diffley, J. F. X., and Nasmyth, K. (1995) Curr. Biol. 5, 1257-1269[Medline] [Order article via Infotrieve] |
9. | Hennessy, K. M., Lee, A., Chen, E., and Botstein, D. (1991) Genes Dev. 5, 958-969[Abstract] |
10. | Nasmyth, K., and Nurse, P. (1981) Mol. Gen. Genet. 182, 119-124[Medline] [Order article via Infotrieve] |
11. | Miyake, S., Okishio, N., Samejima, I., Hiraoka, Y., Toda, T., Saitoh, I., and Yanagida, M. (1993) Mol. Biol. Cell 4, 1003-1015[Abstract] |
12. |
Moir, D.,
Stewart, S.,
Osmond, B.,
and Botstein, D.
(1982)
Genetics
100,
547-563 |
13. |
Maine, G. T.,
Sinha, P.,
and Tye, B. K.
(1984)
Genetics
106,
365-385 |
14. | Yan, H., Gibson, S., and Tye, B. K. (1991) Genes Dev. 5, 944-957[Abstract] |
15. | Takahashi, K., Yamada, H., and Yanagida, M. (1994) Mol. Biol. Cell 5, 1145-1158[Abstract] |
16. | Gibson, S., Surosky, R., and Tye, B. K. (1990) Mol. Cell. Biol. 10, 5707-5720[Medline] [Order article via Infotrieve] |
17. | Hennessy, K., Clark, C., and Botstein, D. (1990) Genes Dev. 4, 2252-2263[Abstract] |
18. | Coxon, A., Maundrell, K., and Kearsey, S. (1992) Nucleic Acids Res. 20, 5571-5577[Abstract] |
19. | Whitbread, L., and Dalton, S. (1995) Gene (Amst.) 155, 113-117[CrossRef][Medline] [Order article via Infotrieve] |
20. | Dalton, S., and Whitbread, L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2514-2518[Abstract] |
21. | Chong, J. P., Thommes, P., and Blow, J. J. (1996) Trends Biochem. Sci. 21, 102-106[CrossRef][Medline] [Order article via Infotrieve] |
22. | Kearsey, S. E., and Labib, K. (1998) Biochim. Biophys. Acta 1398, 113-136[Medline] [Order article via Infotrieve] |
23. | Tye, B. K. (1999) Annu. Rev. Biochem. 68, 649-686[CrossRef][Medline] [Order article via Infotrieve] |
24. | Yan, Y., Merchant, A. M., and Tye, B. K. (1993) Genes Dev. 7, 2149-2160[Abstract] |
25. |
Labib, K.,
Tercero, J. A.,
and Diffley, J. F. X.
(2000)
Science
288,
1643-1647 |
26. |
Todorov, I. T.,
Pepperkok, R.,
Philipova, R. N.,
Kearsey, S. E.,
Ansorge, W.,
and Werner, D.
(1994)
J. Cell Sci.
107,
253-265 |
27. | Kimura, H., Nozaki, N., and Sugimoto, K. (1994) EMBO J. 13, 4311-4320[Abstract] |
28. |
Fujita, M.,
Kiyono, T.,
Hayashi, Y.,
and Ishibashi, M.
(1996)
J. Biol. Chem.
271,
4349-4354 |
29. | Madine, M. A., Khoo, C. Y., Mills, A. D., and Laskey, R. A. (1995) Nature 375, 421-424[CrossRef][Medline] [Order article via Infotrieve] |
30. | Kubota, Y., Mimura, S., Nishimoto, S., Takisawa, H., and Nojima, H. (1995) Cell 81, 601-609[Medline] [Order article via Infotrieve] |
31. | Treisman, J. E., Follette, P. J., O'Farrell, P. H., and Rubin, G. M. (1995) Genes Dev. 9, 1709-1715[Abstract] |
32. | Feger, G., Vaessin, H., Su, T. T., Wolff, E., Jan, L. Y., and Yan, Y. N. (1995) EMBO J. 14, 5387-5398[Abstract] |
33. |
Iizuka, M.,
and Stillman, B.
(1999)
J. Biol. Chem.
274,
23027-23034 |
34. | Borrow, J., Stanton, V. P., Andresen, J. M., Becher, R., Behm, F. G., Chaganti, R. S. K., Civin, C. I., Disteche, C., Dube, I., Frischauf, A. M., Horsman, D., Mitelman, F., Volinia, S., Watmore, A. E., and Housman, D. E. (1996) Nat. Genet. 14, 33-41[Medline] [Order article via Infotrieve] |
35. |
Champagne, N.,
Bertos, N. R.,
Pelletier, N.,
Wang, A. H.,
Vezmar, M.,
Yang, Y.,
Heng, H. H.,
and Yang, X.-J.
(1999)
J. Biol. Chem.
274,
28528-28536 |
36. | Kamine, J., Elangovan, B., Subramanian, T., Coleman, D., and Chinnadurai, G. (1996) Virology 216, 357-366[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Hilfiker, A.,
Hilfiker-Kleiner, D.,
Pannuti, A.,
and Lucchesi, J. C.
(1997)
EMBO J.
16,
2054-2060 |
38. | Reifsnyder, C., Lowell, J., Clarke, A., and Pillus, L. (1996) Nat. Genet. 14, 42-49[Medline] [Order article via Infotrieve] |
39. |
Ehrenhofer-Murray, A. E.,
Rivier, D. H.,
and Rine, J.
(1997)
Genetics
145,
923-934 |
40. |
Smith, E. R.,
Eisen, A.,
Gu, W.,
Sattah, M.,
Pannuti, A.,
Zhou, J.,
Cook, R. G.,
Lucchesi, J. C.,
and Allis, C. D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
3561-3565 |
41. | Mulhard, D., Hunter, R., and Parker, R. (1992) Yeast 8, 79-82[Medline] [Order article via Infotrieve] |
42. | Vidal, M. (1997) in The Yeast Two-hybrid System (Bartel, P. , and Fields, S., eds) , pp. 109-147, Oxford University Press, New York |
43. |
Asano, M.,
and Wharton, R. P.
(1999)
EMBO J.
18,
2435-2448 |
44. | Ohtani, K., Tsujimoto, A., Ikeda, M., and Nakamura, M. (1998) Oncogene 17, 1777-1785[CrossRef][Medline] [Order article via Infotrieve] |
45. |
He, T.-C.,
Zhou, S.,
da Costa, L. T., Yu, J.,
Kinzler, K. W.,
and Vogelstein, B.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2509-2514 |
46. | Nevins, J. R., DeGregori, J., Jakoi, L., and Leone, G. (1997) Methods Enzymol. 283, 205-219[CrossRef][Medline] [Order article via Infotrieve] |
47. | Reynolds, A., Lundblad, V., Dorris, D., and Keaveney, M. (1997) in Current Protocols in Molecular Biology (Ausubel, F. M. , Brent, R. , Kingston, R. E. , Moore, D. D. , Seidman, J. G. , Smith, J. A. , Struhl, K. , and Benson Chanda, V., eds) , pp. 13.6.2-13.6.3, John Wiley & Sons, Inc., New York |
48. |
Sharma, M.,
Zarnegar, M.,
Li, X.,
Lim, B.,
and Sun, Z.
(2000)
J. Biol. Chem.
275,
35200-35208 |
49. | Richter, A., Baack, M., Holthoff, H. P., Ritzi, M., and Knippers, R. (1998) Biol. Chem. 379, 1181-1187[Medline] [Order article via Infotrieve] |
50. | Berg, J. M., and Shi, Y. (1996) Science 271, 1081-1085[Abstract] |
51. | Baldarelli, R. M., Mahoney, P. A., Salas, F., Gustavson, E., Boyer, P. D., Chang, M. F., Roark, M., and Lengyel, J. A. (1988) Dev. Biol. 125, 85-95[Medline] [Order article via Infotrieve] |
52. | Ruiz-i-Altaba, A., Perry-O'Keefe, H., and Melton, D. A. (1987) EMBO J. 6, 3065-3070[Abstract] |
53. | Morishita, K., Parker, D. S., Mucenski, M. L., Jenkins, N. A., Copeland, N. G., and Ihle, J. N. (1988) Cell 54, 831-840[Medline] [Order article via Infotrieve] |
54. |
Ishimi, Y.,
Komamura, Y.,
You, Z.,
and Kimura, H.
(1998)
J. Biol. Chem.
273,
8369-8375 |
55. | Koonin, E. V. (1993) Nucleic Acids Res. 21, 2541-2547[Abstract] |
56. |
Vidal, M.,
Brachmann, R. K.,
Fattaey, A.,
Harlow, E.,
and Boeke, J. D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
10315-10320 |
57. | Allfrey, V., Faulkner, R. M., and Mirskey, A. E. (1964) Proc. Natl. Acad. Sci. U. S. A. 51, 786-794[Medline] [Order article via Infotrieve] |
58. |
Kingston, R. E.,
and Narlikar, G. J.
(1999)
Genes Dev.
13,
2339-2352 |
59. |
Clarke, A. S.,
Lowell, J. E.,
Jacobson, S. J.,
and Pillus, L.
(1999)
Mol. Cell. Biol.
19,
2515-2526 |
60. |
Yamamoto, T.,
and Horikoshi, M.
(1997)
J. Biol. Chem.
272,
30595-30598 |
61. |
McMurry, M. T.,
and Krangel, M. S.
(2000)
Science
287,
495-498 |
62. | Ikura, T., Ogryzko, V. V., Grigoriev, M., Groisman, R., Wang, J., Horikoshi, M., Scully, R., Qin, J., and Nakatani, Y. (2000) Cell 102, 463-473[Medline] [Order article via Infotrieve] |
63. | Weiss, G., and Puschendorf, B. (1988) FEBS Lett. 238, 205-210[CrossRef][Medline] [Order article via Infotrieve] |
64. | Talasz, H., Weiss, G., and Puschendorf, B. (1990) FEBS Lett. 264, 141-144[CrossRef][Medline] [Order article via Infotrieve] |
65. | Belyaev, N. D., Keohane, A. M., and Turner, B. M. (1996) Exp. Cell Res. 225, 277-285[CrossRef][Medline] [Order article via Infotrieve] |
66. | Alexiadis, V., Halmer, L., and Gruss, C. (1997) Chromosoma 105, 324-331[CrossRef][Medline] [Order article via Infotrieve] |
67. | Aparicio, O. M., Weinstein, D. M., and Bell, S. P. (1997) Cell 91, 59-69[CrossRef][Medline] [Order article via Infotrieve] |
68. | Aoki, F., and Schultz, R. M. (1999) Zygote 7, 165-172[CrossRef][Medline] [Order article via Infotrieve] |
69. |
Leone, G.,
DeGregori, J.,
Yan, Z.,
Jakoi, L.,
Ishida, S.,
Williams, R. S.,
and Nevins, J. R.
(1998)
Genes Dev.
12,
2120-2130 |