Báo cáo Y học: Distinct parts of minichromosome maintenance protein 2 associate with histone H3/H4 and RNA polymerase II holoenzyme pptx

Distinct parts of minichromosome maintenance protein 2 associate
with histone H3/H4 and RNA polymerase II holoenzyme
Linda Holland, Michael Downey, Xiaomin Song*, Laura Gauthier, Patricia Bell-Rogers
and Krassimir Yankulov
Department of Molecular Biology and Genetics, University of Guelph, Ontario Canada
Minichromosome maintenance (MCM) proteins are part of
the replication licensing factor (RLF-M), which limits the
initiation of DNA replication to once per cell cycle. We have
previously reported that higher order complexes of mam-
malian pol II and general pol II transcription factors,
referred to as pol II holoenzyme, also contain MCM pro-
teins. In the present study we have analyzed in detail the
interaction between MCM2 and pol II holoenzyme. N- and
C- terminal deletions were introduced into epitope-tagged
MCM2 and the truncated proteins were transiently
expressed in 293 cells. Affinity chromatography was used to
purify RNA pol II holoenzyme and histone binding MCM
complexes. We found that amino acids 168–230 of MCM2
are required for its binding to pol II holoenzyme in vivo.We
also showed that bacterially expressed amino acids 169–212
of MCM2 associate with pol II and several general tran-
scription factors in vitro. Point mutations within the 169–212
domain of MCM2 disrupted its interaction with pol II
holoenzyme both in vitro and in vivo.Thisregionisdistinct
from the previously characterized histone H3 binding
domain of MCM2.
Keywords: MCM2; RNA polymerase II holoenzyme; his-
tone.
Large protein complexes, which contain RNA polymerase
II as well as the general pol II transcription factors (GTFs)
TFII A, B, D, E, F, and H [1] and other proteins have been

isolated from yeast, mammalian and amphibian cells [2–8].
TheyarereferredtoaspolIIÔholoenzymeÕ. Some of the
components of pol II holoenzyme make direct or indirect
contacts with the C-terminal domain (CTD) of the largest
subunit of pol II. Antibodies against the CTD disrupt the
yeast holoenzyme into core pol II and a mediator subcom-
plex, which contains the SRB and MED proteins [7–9]. A
similar treatment of pol II holoenzyme from HeLa cells also
disrupts its interaction with several of the GTFs [6]. In
higher eukaryotes the CTD mediates the interaction with
complexes that contain homologues of the yeast SRB and
MEDproteinssuchasSMCC[10]orNAT[11].Itis
believed that pol II holoenzyme is a functionally significant
complex, which is responsible for transactivator-stimulated
transcription in vivo. It has been shown that the srb4 and
srb6 genes are essential for expression of most mRNAs in
budding yeast [12]. Other holoenzyme components such as
SRB 2, 5, 7–11, SWI/SNF proteins, SIN4, RGR1, MED2,
MED9/CSE2, MED10/NUT2, MED11, GAL11, PGD1
and ROX3 [7,9,13–16] are not essential for transcription of
most genes but do contribute to the response to transacti-
vators and repressors (reviewed in [17,18]). In addition to its
role in the response to transcriptional regulators, pol II
holoenzyme may be involved in integrating transcription
with RNA processing, DNA repair and replication. In
support of this idea, the DNA repair factors DNA pol e,
XPC, XPF, XPG, Ku, RAD51 [3], BRCA1 [19]; RNA
helicase A [20]; the replication factors RP-A, RP-C [3] and
MCM proteins [6]; and the cleavage/polyadenylation
factors CPSF and CstF [21] have been identified in

mammalian pol II holoenzyme preparations. There are
significant differences in the composition of pol II holoen-
zymes that have been purified by different procedures
indicating that this complex is capable of interacting with a
variety of proteins and that there might be multiple forms of
pol II holoenzyme in vivo [22].
MCM proteins, previously characterized as components
of the replication licensing factor M (RLF-M) [23–26], were
also found to associate with pol II holoenzyme in higher
eukaryotes [6]. It is believed that RLF-M is acting to limit
replication of genomic DNA to a single round per cell cycle
[27]. As predicted by the licensing model, most MCMs are
released from chromatin during S phase and re-associate at
the end of mitosis [28–33]. In addition to promoting
initiation of DNA replication, MCMs also seem to function
in replication fork movement [28,34,35]. The MCM4,6,7
subcomplex possesses DNA helicase activity [35–38], which
has been implicated in both initiation and fork movement.
In addition, MCM complexes bind with high affinity to core
histone H3/H4 dimers [39,40] and to HBO1 [41], via distinct
domains in the N-terminus of MCM2 indicating a possible
Correspondence to K. Yankulov, Department of Molecular Biology
and Genetics, University of Guelph, Guelph, Ontario N1G 2W1,
Canada. Fax: + 1 519 8372075, Tel.: + 1 519 8244120, ext. 6466,
E-mail:
Abbreviations: MCM, minichromosome maintenance; CTD,
carboxy-terminal domain (of the largest subunit of RNA poly-
merase II); GTF, general transcription factor; SMCC, SRB/
MED-containing cofactor; NAT, negative regulator of activated
transcription; RLF-M, replication licensing factor M; TBP, TATA

box binding protein; HBO1, histone acetyltransferase binding to
ORC; ORC, origin recognition complex; FCS, fetal calf serum;
GST, glutathione S-transferase; TFIIS, transcription factor II S.
*Present address: Pharmacia Corporation, AA215/AA2C,
700 Chesterfield Parkway, Chesterfield, MO 63198, USA.
(Received 11 June 2002, revised 26 August 2002,
accepted 30 August 2002)
Eur. J. Biochem. 269, 5192–5202 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03224.x
chromatin remodeling function. MCM2, but not other
MCM proteins, also interacts with cdc6, a component of the
replication preinitiation complex [42]. The significance of
these protein interactions and the precise biochemical role of
MCMs in regulating DNA replication remain unclear.
There are certain indications that MCMs might be
involved in pol II transcription in higher eukaryotes. We
have shown that antibodies against MCM2 inhibit pol II,
but not pol III, transcription from injected template
plasmids in Xenopus oocytes [6]. Two other studies have
demonstrated that an interaction between MCM5 and
the activation domain of Stat1a is essential for the
expression of IFN-c responsive genes [43,44]. On the
other hand, recruitment of pol II holoenzyme to origins
of replication via GAL11 or TBP significantly stimulates
replication of minichromosomes in S. cerevisiae [45],
suggesting a possible role of pol II holoenzyme in
DNA replication.
In this paper we have analyzed the interaction between
human MCM2 (also called BM28) and pol II holoenzyme.
We report that MCM2 binds to pol II holoenzyme via a
sequence in its N-terminal domain. This region is positioned

between the site of interaction with histone H3 and the
putative HBO1 binding site.
MATERIALS AND METHODS
Plasmids
All plasmids for expression of recombinant human
MCM2 encode N-terminally FLAG-tagged polypeptides.
pFLAG-MCM2(FL) contains the EcoRI fragment of
pBSBM28 ([46], EMBL accession no. P49736), cloned
into the EcoRI site of pFLAG-CMV-2 (Sigma). This
plasmid and all its derivatives encode MDYKDDDDK
LAAANSAESSESFT followed by different MCM2 frag-
ments. pFLAG-MCM2(1–197), pFLAG-MCM2(1–247),
and pFLAG-MCM2(1–511) were generated by deleting
the Sal I, the Bgl II, and the EcoRV fragments from
pFLAG-MCM2(FL), respectively. pFLAG-MCM2(1–
167) was generated by subcloning the EcoR1-Dra III
fragment of pBSBM28 into EcoRI-Sma I linearized
pFLAG-CMV-2. pFLAG-MCM2(1–230) was generated
by deleting the BsaAI-Sma I fragment from pFLAG-
MCM2(1–247). pFLAG-MCM2(198–892) contains the
Sal I fragment of pBSBM28 [46] cloned into the Sal I
site of pFLAG-CMV-2 and encodes MDYKDDDDK
LAAANSSIDLISVPV followed by amino acids 198–892.
pFLAG-MCM2(345–892) contains the Fse I-SmaIfrag-
ment of pBSBM28 [46] cloned into Not I-Sma I linearized
pFLAG-CMV-2 and encodes MDYKDDDDKLA fol-
lowed by amino acids 345–892 of MCM2. All expression
plasmids were purified by anion exchange (Qiagen) prior
to transfection. pGEX-MCM2(169–212) contains the
sequence encoding amino acids 169–212 of MCM2

attached in frame to GST. Site-directed mutagenesis of
the pGEX-MCM2(169–212) and pFLAG-MCM2(1–230)
plasmids was conducted using the Quikchange site-direc-
ted mutagenesis kit (Stratagene). 5¢-CCGCTTCAA
GAACTTCCCGGGCACTCACGTCAC-3¢ was used as
a primer to introduce changes from LR to PG at
positions 192/193, and 5¢-GCCACGGCCACAACGAG
CTCAAGGAGCGCATCAGC-3¢ was used to introduce
changes from VF to EL at positions 203/204. Point
mutations were confirmed by nucleotide sequencing.
Antibodies
Anti-(Pol II CTD) (8WG16) [47], anti-(BM28-N) directed
towards the N-terminus of MCM2 [32], anti-TBP [6], anti-
TFIIB [6], anti-CPSFp160 [6], and anti-(Xenopus ORC2)
[48] were described previously. Anti-MCM was generated
against a highly conserved peptide sequence VVCI
DEFDKMSDMDRTA, which is shared between all
MCM proteins [49]. The antibody was affinity purified on
antigenic peptide coupled to Affigel-10 (Bio-Rad). Anti-
p62(TFIIH) was raised against full length human p62
expressed in E. coli. The anti-FLAG antibody M2 was
purchased from Sigma. The anti-CycC was from Santa
Cruz Biotechnology.
Expression of recombinant MCM2 proteins in human
embryonic kidney fibroblast cells (293)
HEK 293 cells were grown in 15 cm plates (Costar) to 50%
confluency in DMEM medium supplemented with 10%
fetal bovine serum and antibiotics (100 unitsÆmL
)1
penicillin

and 100 lgÆmL
)1
streptomycin). Each plate was transfected
with 20 lg of pFLAG-CMV-MCM2 expression plasmid
plus 20 lg of carrier plasmid (pBS) using calcium phos-
phate-precipitation. Transfection efficiency was between
30% and 50% as monitored by the expression of green
fluorescent protein from pEGFP-C2 (Clontech).
Preparation of whole-cell extract
Cells were harvested 36–48 h after transfection and whole-
cell extract was prepared by lysis in hypotonic buffer and
0.41
M
(NH
4
)
2
SO
4
extraction as described previously [6].
Prior to chromatography, each extract was buffer
exchanged in a 10DG column (Bio-Rad) to chromato-
graphy buffer (CB) (10 m
M
Hepes 7.9, 0.2 m
M
EDTA,
0.2 m
M
EGTA, 5 m

M
2-glycerophosphate, 1 m
M
Na
3
VO
4
,
1m
M
NaF, 1 m
M
benzamidine, 1 m
M
dithiothreitol, 50 l
M
ZnCl
2
,1lgofpepstatinÆmL
)1
,1lg of leupeptinÆmL
)1
,
2 lg of aprotininÆmL
)1
, 12% glycerol, 0.05% NP-40) plus
50 m
M
NaCl and clarified by centrifugation for 15 min at
21 000 g. Protein concentration of the extracts after dialysis

was 5–10 mgÆmL
)1
.
GST-TFIIS affinity chromatography
RNA polymerase II holoenzyme was purified by GST-
TFIIS affinity chromatography as described previously [6].
Briefly, Glutathione S-transferase (GST), and GST-TFIIS
(residues 1–301 of mouse transcription factor IIS) were
expressed in E. coli BL21(LysS) and immobilized on
Glutathione Sepharose 4B (Pharmacia) at 10 mgÆmL
)1
.
Mini-columns containing 100 lL of beads were prepared.
Extracts from four tissue culture plates were passed through
a GST column followed by a GST-TFIIS column. Each
column was washed twice with 1 mL of CB plus 50 m
M
NaCl, then five times with 100 lLofCBplus50m
M
NaCl,
eluted four times with 100 lLCBplus0.325
M
NaCl and
then four times with 100 lL with CB plus 1
M
NaCl. Final
100 lL wash and eluate fractions were precipitated in
Ó FEBS 2002 MCM2 binds to pol II holoenzyme (Eur. J. Biochem. 269) 5193
0.8 mgÆmL
)1

deoxycholic acid and 20% trichloroacetic acid
and then re-suspended in SDS/PAGE sample loading
buffer.
Histone H3/H4 affinity chromatography
H3/H4 histones were purified from HeLa cell nuclear pellets
following the protocol of Simon and Felsenfeld [50] and
coupled to Affigel-10 (Bio-Rad) at 5 mgÆmL
)1
of resin.
Bovine serum albumin (Fraction V, Sigma) was coupled to
Affigel-10 at a concentration of 5 mgÆmL
)1
. Purification of
MCM proteins on H3/H4-Affigel beads was carried out as
described previously [39] with some modifications. Briefly,
flow-through fractions from the GST-TFIIS columns
(3 mL at 5 mgÆmL
)1
protein) were loaded sequentially to
a BSA-Affigel column (100 lL) followed by a histone
H3/H4 column (100 lL) equilibrated with buffer A (20 m
M
Tris/HCl pH 7.5, 0.5 m
M
EDTA, 1 m
M
dithiothreitol,
0.1 m
M
phenylmethanesulfonyl fluoride, and 10% glycerol)

containing 0.1
M
NaCl. The columns were washed two
timeswith1 mLandfivetimeswith200 lL of buffer A plus
0.1
M
NaCl, and were eluted with 0.5
M
,0.75
M
and 2
M
NaCl in buffer A (1 mL, 600 lL, and 2 mL, respectively).
Wash and eluate fractions were precipitated in 0.8 mgÆmL
)1
deoxycholic acid/20% trichloroacetic acid, then resuspen-
ded in SDS/PAGE sample loading buffer.
GST-MCM2(169–212) affinity chromatography
GST, GST-TFIIS, GST-MCM2(169–212)wt, GST-
MCM2(169–212)L192P/R193G, and GST-MCM2(169–
212)V203E/F204L proteins were expressed in
BL21(LysS)DE3 cells and coupled to glutathione Sepharose
4B (Pharmacia) at 10 mgÆmL
)1
. Each column (250 lL) was
loaded with HeLa whole cell extract (10 mgÆmL
)1
[6]),
columns were extensively washed and eluted with 1
M

NaCl.
Samples were precipitated with 0.8 mgÆmL
)1
deoxycholic
acid and 20% trichloroacetic acid and analyzed by Western
blotting.
Western blotting
Proteins were transferred to Immobilon-P membrane (Mil-
lipore) by semidry electroblotting. Blots were developed by
BM Chemiluminescence Blotting Substrate (Roche) or ECL
Plus (Amersham) with horseradish peroxidase coupled to
secondary antibody (Sigma or Amersham). For quantita-
tion, blots were exposed on an Image Station (Kodak,
440CF) and images were analyzed by Kodak 1D Image
Analysis Software.
Proteomics tools
Multiple sequence analysis was performed by
BLAST
.
Three dimensional structure prediction was carried out by
3
D
-
PSST
( and Swiss-Model
( />Prediction of sites of phosphorylation was by
NETPHOS
2.0
( Hydrophobicity
and charge analysis was performed by

PROTPARAM
( Secondary
structure prediction was by
JPRED
2 (.
uk:8888).
RESULTS
Experimental Strategy
Previous experiments have shown that antibodies against
MCM2 specifically inhibit pol II transcription in Xenopus
oocytes [6]. We decided to search for domain(s) in this
polypeptide that might be responsible for the interaction
between MCM proteins and pol II holoenzyme [6]. FLAG-
tagged human MCM2 deletion mutants (Fig. 1) were
expressed in 293 cells and assayed for their ability to
copurify with pol II holoenzyme or to bind to histones
H3/H4. This approach circumvented problems with bac-
terial expression of MCM2 that we had encountered in the
past (data not shown). Extracts were prepared from
transfected cells and pol II holoenzyme was purified by
affinity chromatography using GST-TFIIS as a ligand [5,6].
We had previously shown that about 2% of the total
endogenous MCM2 in HeLa cell extract copurified with
pol II holoenzyme on GST-TFIIS columns. The flow-
through fractions of the GST-TFIIS chromatography,
which contained the majority of MCM proteins, were
subsequently chromatographed on histone H3/H4-agarose
as described [38–40]. The binding of different MCM2
deletion mutants to GST-TFIIS-Sepharose or H3/H4
histones relative to endogenous MCM2 and other MCM

proteins was analyzed by Western Blotting.
Plasmids encoding for recombinant MCM2 polypeptides
(Fig. 1) were transfected into 293 cells and expression was
Fig. 1. Scheme of constructs for the expression of FLAG-tagged
MCM2 deletion mutants. Human MCM2 DNA sequences encoding
the indicated amino acid residues in the full length protein were cloned
into pFLAG-CMV-2. All sequences contain a N-terminal FLAG tag.
(+) denotes that binding of the expressed protein to pol II holoenzyme
or histone H3/H4 was observed. (–) denotes that no binding was
observed. (–/+) denotes very weak binding.
5194 L. Holland et al. (Eur. J. Biochem. 269) Ó FEBS 2002
allowed to proceed for 36–48 h. Whole cell extracts were
prepared as described previously [6]. Under these conditions
all recombinant MCM2 polypeptides were expressed and
extracted at levels, which were roughly equal to the
endogenous MCM2 with the exception of MCM2(198–
892), which was expressed at somewhat lower levels (Fig. 2
and data not shown).
Binding of MCM deletion mutants to GST-TFIIS beads
GST-TFIIS affinity chromatography was used to purify
RNA pol II holoenzyme and associated MCM proteins
from whole cell extracts. Each extract was passed through a
control column containing GST alone and then loaded on a
GST-TFIIS column. Both columns were washed extensively
with low-salt buffer and eluted with 0.325
M
NaCl and then
with 1
M
NaCl. Western blots of the load, flow-through,

wash, 0.325
M
eluate, and 1
M
eluate fractions are shown in
Fig. 3. As reported previously [5,6], pol II bound to GST-
TFIIS and eluted as two distinct fractions at 0.325
M
NaCl,
which corresponds to pol II holoenzyme, and 1
M
NaCl,
which corresponds to core pol II (Fig. 3, lanes 1–9). The
amounts of pol II in the 0.325
M
NaCl eluates were similar
between different extracts as detected by an antibody
against its largest subunit [5,6]. There were noticeable
differences in the intensity of pol II signal in the 1
M
eluate
probably because of the limiting quantities of antigen in this
fraction that occasionally might be below the sensitivity of
our antibody. Importantly, in the 0.325
M
NaCl eluate from
all extracts we detected comparable amounts of endogenous
Fig. 2. Expression of FLAG-tagged MCM2 deletion mutants in 293
cells. Plasmids encoding N-terminally FLAG-tagged MCM2 deletion
mutants were individually transfected in 293 cells and expressed for

36–48 h. Whole cell extracts were prepared as described in Materials
and methods and 90 lg were loaded per lane. Proteins were separated
on 10% SDS/PAGE gels and analyzed by Western blotting with anti-
FLAG Ig.
Fig. 3. Analysis of binding of FLAG-tagged MCM2 deletion mutants to GST-TFIIS. Affinity columns (100 lL) containing GST (10 mgÆmL
)1
)or
GST-TFIIS (10 mgÆmL
)1
) were loaded in series with 20–40 mg of 293 whole-cell extract, washed extensively and eluted with 0.325
M
NaCl and then
with 1
M
NaCl. 0.33% of the Load (L), flowthrough (FT), and 33% of the final wash (W) and eluate (E) fractions were analyzed by Western
blotting with the indicated antibodies. The figure shows one of three independent experiments (lines a–d) or one of two independent experiments
(lines e–i).
Ó FEBS 2002 MCM2 binds to pol II holoenzyme (Eur. J. Biochem. 269) 5195
MCM2 (Fig. 3, lanes 10–18). Binding of full length FLAG-
tagged MCM2 to GST-TFIIS is shown in Fig. 3, row (a),
lanes 19–27.
Western blotting with anti-FLAG Ig was used to
compare the binding of MCM2 deletion mutants to GST-
TFIIS relative to the endogenous MCM2 (Fig. 3, lanes 10–
18) and to the full length FLAG-tagged MCM2. Binding to
GST-TFIIS was considered positive when signals in the
0.325
M
NaCl eluates (Fig. 3, lanes 16 and 26) were stronger
than the signals in the final wash fractions (Fig. 3, lanes 15

and 25). While N-terminal deletions such as MCM2(198–
892) and MCM2(345–892) (Fig. 3, rows g, h) displayed
deficient association with GST-TFIIS, the C-terminal
deletion mutants MCM2(1–230), MCM2(1–247), and
MCM2(1–511)(Fig. 3, rows d–f) all coeluted with pol II
holoenzyme at comparable levels to that of full length-
MCM2(1–892) (Fig. 3, row a). However, further C-terminal
deletions MCM2(1–197) and MCM2(1–167) caused an
incremental decrease and disappearance of the FLAG
signal in the 0.325
M
eluate (Fig. 3, rows b, c), while
endogenous MCM2 signal in this fraction was similar for all
extracts. These initial results suggested that amino acids
168–230 of MCM2 could be involved in the interaction
between MCM proteins and pol II holoenzyme.
Binding of MCM deletion mutants to H3/H4 dimers
MCM proteins bind to histone H3/H4 dimers in vitro via an
interaction mediated by the N terminus of MCM2 [38–40].
It is possible that the interaction between MCMs and the
pol II holoenzyme is mediated by histones, which could be
recruited to the holoenzyme in a specific or nonspecific
manner. We tested this possibility by analyzing the binding
of the MCM2 mutants to histone H3/H4. The flow-through
fractions of the GST-TFIIS columns were loaded sequen-
tially on BSA-agarose and H3/H4-agarose columns. The
resins were washed with low salt buffer and eluted with 0.5,
0.75, and 2
M
NaCl. Load, flow-through, final wash and

eluate fractions were analyzed by Western blotting.
First we examined the binding of MCMs to the H3/H4
resin using an antibody against the conserved ATP binding
domain of all MCM proteins [49]. This antibody cross-
reacts with many bands in crude extracts; however, in
purified fractions it detects three to five bands, which
correspond to MCM proteins [36,39,51]. In the wash
fraction and the eluates of the H3/H4 columns we detected
three to five bands with the expected mobility of MCM2,
MCM3, MCM4, MCM5 and MCM6 (Fig. 4, lanes 5–7).
These signals were significantly higher than the correspond-
ing background signals from the control BSA-agarose
columns (Fig. 4, lanes 1–4). We also detected recombinant
MCM2(198–892) and MCM2(345–892), which contained
intact ATP binding domain (data not shown). Because this
antibody has a low affinity, we did not observe exactly the
same profile of bands in all eluates possibly because some of
the MCM proteins were present below the threshold of
detection. We did not see any MCMs in the 2
M
NaCl eluate
by this or other anti-MCM Ig (not shown). This observation
is in disagreement with the previously reported histone H3/
H4 chromatography experiments, in which the majority of
Fig. 4. Analysis of binding of FLAG-tagged
MCM2 deletion mutants to H3/H4-agarose.
Affinity columns (100 lL) containing bovine
serum albumin (BSA, 5 mgÆmL
)1
) and histone

H3/H4 (5 mgÆmL
)1
) were loaded in series with
15–30 mg of GST-TFIIS column flow-
through, washed and eluted with buffer A
containing 0.5
M
NaCl, buffer A containing
0.75
M
NaCl, and buffer A containing 2
M
NaCl. 0.33% of the Load (L), flowthrough
(FT), wash (W) and eluate (E) fractions were
analyzed by Western blotting with the indi-
cated antibodies. In rows a, e, f, g, and h, we
pooled all wash fractions and eluates and
loaded 33% of each per lane, respectively. In
rows b, c, and d, we show 33% of final
washand33%ofthepooled0.5
M
and 0.75
M
eluate. The figure shows one of three inde-
pendent experiments (lines a–d) or one of two
independent experiments (lines e–i).
5196 L. Holland et al. (Eur. J. Biochem. 269) Ó FEBS 2002
MCMs were found in the 2
M
salt eluate [38–40]. We do not

understand this discrepancy. Nonetheless, our histone
H3/H4 resin specifically retained MCM proteins as reported
in [38–40] and was considered adequate for further analyses.
Next we examined the binding of the MCM2 deletion
mutants to H3/H4 relative to the endogenous MCM2 and
to the full length FLAG-tagged MCM2. Significant
amounts of endogenous MCM2 were found in all histone
H3/H4 eluates while no MCM2 was detected in the
corresponding eluates from the control BSA-agarose col-
umn (Fig. 4, lanes 8–14). In agreement with previous
reports [38–40], the N-terminal deletion mutants
MCM2(198–892) and MCM2(345–892) did not associate
with histone H3/H4 (Fig. 4, lanes 15–21, rows g and h). All
other recombinant polypeptides closely resembled the
elution pattern of the endogenous MCM2 (Fig. 4, lanes
15–21). Importantly, the MCM2(1–167) and MCM2(1–
197), which did not bind to GST-TFIIS, bound strongly to
histones (Fig. 4, lanes 15–21, rows b and c).
This second set of experiments clearly demonstrated that
the sequence of MCM2, which confers association with
pol II holoenzyme (amino acids 168–230, Fig. 3) is distinct
from the sequence, which is required for its association with
histone H3/H4 (amino acids 1–167, Fig. 4) [38–40].
Binding of pol II holoenzyme to GST-MCM2(169–212)
We tested the possibility that amino acids 168–230 of
MCM2 were required for binding to pol II holoenzyme by a
different procedure. We expressed this peptide as a GST-
fusion protein and used it in affinity chromatography
experiments with HeLa cell extract. Because the C-terminus
of 168–230 contains the peptide VNYEDLA, which is part

of the reported HBO1 binding site [41], we decided to
further truncate the C-terminus of this sequence to produce
GST-MCM2(169–212) fusion protein. The protein was
coupled to glutathione beads and HeLa cell extract was
passed in parallel through GST, GST-TFIIS and GST-
MCM2(169–212) beads, the beads were washed extensively
withCBbufferandelutedwith1
M
NaCl. The load,
flowthrough, final wash and eluate fractions were analyzed
by Western blotting (Fig. 5). In the eluates of both GST-
TFIIS and GST-MCM2(169–212) columns we detected the
largest subunit of pol II together with subunits of the
general transcription factors TFIID (TBP), TFIIH(p62)
and TFIIB. Other components of pol II holoenzyme such
as CycC(SRB10) and CPSF(p160) were present at signifi-
cantly lower levels in the GST-MCM2(169–212) eluates
relative to the GST-TFIIS eluates. A component of the
origin recognition complex (ORC2), which associates with
MCM proteins at origins of replication [52], was not
detected in the eluates of both columns indicating that the
observed signals are not a consequence of contamination by
extract or chromatin. In all Western blots very little or no
signal from all antigens was observed in the final wash
fractions and control GST eluates.
In summary, we observed that MCM2(169–212) was
binding pol II, TFIID, TFIIH and TFIIB with similar
(albeit lesser) efficiency as compared to a bona fide
holoenzyme binding ligand, TFIIS [5,6]. This data is
consistent with the idea that amino acids 169–212 of

MCM2 are binding to some component(s) of RNA
polymerase II holoenzyme.
Point mutations in the MCM2(169–212) domain disrupt
the binding of pol II holoenzyme
in vitro
and
in vivo
If the MCM2(169–212) domain is required for binding to
pol II holoenzyme, then mutations in this region should
disrupt the interactions, which were described in Figs 3 and
5. To test this hypothesis, we substituted conserved hydro-
philic and hydrophobic residues in GST-MCM2(169–212)
as shown in Fig. 6A. GST, GST-MCM2(169–212)V203E/
F204L, GST-MCM2(169–212)L192P/R193G, and GST-
MCM2(169–212)wt were coupled to beads at 10 mgÆmL
)1
(data not shown) and assayed for their ability to pull down
components of pol II holoenzyme from HeLa extract. Each
column was loaded with 100 mg of HeLa whole cell extract,
washed extensively with CB, and eluted with 1
M
NaCl.
Flowthrough, final wash and eluate fractions were analyzed
by Western blotting with antibodies against pol II and
several general transcription factors (Fig. 6B). Consistent
with our previous experiment (Fig. 5), considerable
amounts of pol II, TFIID(TBP), TFIIH(p62) and TFIIB
were detected in the GST-MCM2(169–212)wt eluate
(Fig. 6B, lane 12), while little to no signal was detected in
the wash fractions or in the GST control eluate (Fig. 6B,

lane 3). GST-MCM2(169–212)L192P/R193G retained sim-
ilar or slightly lower amounts of pol II, TBP, TFIIH(p62)
and TFIIB (Fig. 6B, lane 9) relative to GST-MCM2(169–
212)wt. A dramatic decrease in the signals from all peptides
was observed in the eluate of the GST-MCM2(169–
212)V203E/F204L column (Fig. 6B, lane 6). These results
Fig. 5. GST-MCM2(169–212) chromatography. Affinity columns
(250 lL) containing GST, GST-MCM2(169–212), or GST-TFIIS
(each at 10 mgÆmL
)1
)wereloadedinparallelwith50mgofHeLacell
extract, washed extensively and eluted with 1
M
NaCl. 0.33% of the
Load, flow-through, and 40% of the final wash and eluate fractions
were analyzed by Western blotting with the indicated antibodies. The
figure shows one of two independent experiments.
Ó FEBS 2002 MCM2 binds to pol II holoenzyme (Eur. J. Biochem. 269) 5197
clearly indicated that the V203E/F204L substitution dis-
rupted the ability of the MCM2(169–212) peptide to bind
pol II and general transcription factors in vitro.
Next we tested if the same mutations would interfere with
the binding of MCM2 to pol II holoenzyme in vivo.We
introduced the V203E/F204L and L192P/R193G substitu-
tions into the pFLAG-MCM2(1–230) plasmid. FLAG-
MCM2(1–230) was the smallest stable deletion mutant,
which contained the 169–212 region and retained full
capacity to copurify with pol II holoenzyme (Fig. 3). The
wild type and mutant proteins were expressed in 293 cells
and extracted at comparable levels (Fig. 7A). Each extract

was assayed by GST-TFIIS affinity chromatography. Load,
flowthrough, wash and 0.325
M
NaCl eluate fractions were
analyzed by Western blotting with anti-CTD Ig, and anti-
MCM2 Ig, which recognized equally well the endogenous
MCM2 and all MCM2(1–230) recombinant peptides
(Fig. 7A). As observed in our previous experiment (Fig. 3)
the largest subunit of pol II and endogenous MCM2
coeluted in the 0.325
M
NaCl eluate of the GST-TFIIS
column. The amounts of these two proteins in this fraction
were similar for all extracts (Fig. 7B, lanes 7 and 14). We
then compared the copurification of each MCM2(1–230)
mutant relative to endogenous MCM2 and to the
MCM2(1–230)wt control (Fig. 7B, row a). MCM2(1–
230)L192P/R193G (Fig. 7B, row c, lane 14) displayed
somewhat deficient association with GST-TFIIS, whereas
MCM2(1–230)V203E/F203L was almost completely absent
from the pol II holoenzyme fraction (Fig. 7B, row b, lane
14). The relative abundance of the wild type and mutant
MCM2(1–230) peptides in the eluates from the GST-TFIIS
columns was confirmed by Western blot with anti-FLAG
antibodies (data not shown). These results are in agreement
with the results obtained with GST-fusion proteins in vitro
(Fig. 6). We conclude that the V203E/F203L substitution
disrupts the association of MCM2 with pol II holoenzyme
both in vitro and in vivo. The experiments presented in
Figs 6 and 7 further verify the importance of amino acids

169–212 to the interaction between MCM2 and RNA pol II
holoenzyme.
DISCUSSION
MCM2 interacts with pol II holoenzyme
We have previously shown that several MCM proteins
copurified with pol II holoenzyme preparations from
human and Xenopus cells [6]. In this paper we show that
a likely site, which mediates this interaction is amino acids
169–212 of MCM2. Our results demonstrate that in vivo
expressed epitope-tagged MCM2 deletion mutants bind to
GST-TFIIS and elute in the pol II holoenzyme fraction
as shown before [5,6] only if they contain amino acids
168–230 (Fig. 3). N- and C-terminal deletions within this
region significantly decreased, but did not completely
abolish binding to GST-TFIIS (Fig. 3, lines c and g).
The C-terminal deletions MCM2(1–167) and MCM2(1–
197) retained their ability to associate with histones H3/
H4 (Fig. 4, lines b and c) as previously reported [38–40].
Therefore, it is unlikely that their deficiency in binding
pol II holoenzyme is a result of misfolding or inactivation.
We do not have a reliable assay to test the functional
status of MCM2(198–892) and MCM2(345–892) (Figs 3
and 4) and other N-terminal deletions beyond amino acid
345 (not shown), which did not bind to GST-TFIIS or
histone H3/H4.
In a separate set of experiments we show that a GST-
MCM2(169–212) ligand binds pol II and several previously
characterized components of RNA polymerase II holoen-
zyme in vitro with similar efficiency as compared to GST-
TFIIS (Fig. 5). These experiments imply that MCM2

peptides might be recruited to the holoenzyme independ-
ently of whether they are in a complex with other MCM
proteins or not. Furthermore, a previous study [53] indica-
tedthattheinteractionbetweenMCM2andMCM4,6,7is
located in the C-terminus of MCM2. Previously character-
ized complexes of MCM2 such as RLF-M and
MCM2,4,6,7 [51,54,55] consist of single molecules of the
six or four MCM proteins, respectively. It is therefore
unlikely that the deletion mutants were recruited to GST–
TFIIS via interactions with complexes that already contain
endogenous MCM2.
Fig. 6. Point mutations in the MCM2(169–212) domain interfere with
the binding of pol II and general transcription factors in vitro. (A) The
indicated amino acid substitutions were introduced into the GST-
MCM2(169–212) peptide by site-directed mutagenesis. (B) Affinity
columns (250 lL) containing GST, GST-MCM2(169–212)V203E/
F204L, GST-MCM2(169–212)L192P/R193G, or GST-MCM2(169–
212)wt (each at 10 mgÆmL
)1
) were loaded with 100 mg of HeLa whole
cell extract, washed extensively and eluted with 1
M
NaCl. 0.33% of the
Flowthrough (FT) and 40% of each final wash (W) and eluate (E)
fractions were analyzed by Western blotting with the indicated anti-
bodies. The figure shows one of two independent experiments.
5198 L. Holland et al. (Eur. J. Biochem. 269) Ó FEBS 2002
The idea that MCM2(169–212) is a site for interaction
with pol II holoenzyme is significantly strengthened by the
observation that a double amino acid substitution (V203E/

F204L) within this region disrupted the association of this
domain with pol II holoenzyme both in vitro and in vivo
(Figs 6 and 7). Taken together, our results strongly suggest
that amino acids 169–212, which are not required for the
interaction of MCM2 with histones H3/H4 [39,40] (Fig. 4)
or HBO1 [41], are involved in the interaction with some
component(s) of pol II holoenzyme. We propose that three
different sites of MCM2 are involved in independent
interactions with histone H3/H4, pol II holoenzyme and
HBO1asshowninFig.8.
In light of our previous study [6] the most likely
explanation of our observations is that MCM2(169–212)
associates with some component(s) of pol II holoenzyme
directly or via a bridging interaction. The identity of this
component is not known. We have shown that antibod-
ies against the carboxyterminal domain of the largest
subunit of pol II (CTD) disrupt the interaction between
pol II holoenzyme and MCM proteins [6]. In addition,
MCMs, CPSF, CstF, and TFIIH bound to recombinant
CTD [6]. It is possible that the partner of the
MCM2(169–212) peptide in pol II holoenzyme is within
one of these three factors or it is the CTD itself,
however, other components of the pol II holoenzyme
should also be considered.
Characteristics of the MCM2(169–212) sequence
The MCM2(169–212) sequence displays no obvious fea-
tures that might suggest possible function (See Fig. 8). It has
a pI of 9.18 and evenly distributed positively and negatively
charged residues. Jpred2 indicates two stretches of potential
helices, however, no homology to previously characterized

protein–protein interaction domains and no obvious simi-
larity with known 3D structures as determined by
SWISS
-
MODEL
and 3
D
-
PSSM
were detected. These analyses give us
little hint about the nature of this domain and the possible
mechanism of interaction with potential target peptides.
NETPHOS
2.0 indicated three high score phosphorylation sites
in the putative pol II holoenzyme binding sequence of
MCM2 (Fig. 8). It is conceivable that interaction between
MCMs and holoenzyme is regulated by phosphorylation at
the predicted serine residues, but the significance of these
residues is yet to be established.
The 169–212 amino acid sequence of human MCM2 has
highly homologous counterparts in mouse, Xenopus,and
Drosophila (Fig. 8), suggesting that a similar interaction
between MCM2 and pol II holoenzyme might be taking
place in these organisms. The homology with the cognate
MCM2 regions in S. pombe, C. elegans and S. cerevisiae is
56, 54 and 46%, respectively, with several highly conserved
hydrophobic residues, which are identical in all species
(Fig. 8). The sequence similarities do not provide clues as to
Fig. 7. Point mutations in the MCM2(169–212) domain diminish binding of pol II holoenzyme in vivo. (A) The amino acid substitutions, shown in
Fig. 6A, were introduced into the pFLAG-MCM2(1–230) plasmid by site directed mutagenesis. N-Terminally flag-tagged full-length MCM2(1–

892) (lanes 1 and 5), MCM2(1–230)V203E/F204L (lanes 2 and 6), MCM2(1–230)L192P/R193G (lanes 3 and 7), and MCM2(1–230)wt (lanes 4 and
8) were expressed in 293 cells and whole cell extracts were prepared and 110 lg were loaded per lane. Proteins were resolved on 10% SDS/PAGE
gels and analyzed by Western blotting with anti-FLAG (lanes 1–4) and anti-MCM2 (lanes 5–8) Ig. eMCM2, endogenous MCM2. (B) GST-TFIIS
affinity chromatography was conducted as described in Materials and methods. 0.33% of the Load (L), flowthrough (FT), and 50% of the final
wash (W) and 0.325
M
NaCl eluate (E) fractions were analyzed by Western blotting with antipol II CTD and anti-MCM2 Ig. eMCM2, endogenous
MCM2. The relative net intensities of bands in Lane 14 are plotted and the position of each MCM2(1–230) mutant and its relative eMCM2 control
are indicated. The figure shows one of three independent experiments.
Ó FEBS 2002 MCM2 binds to pol II holoenzyme (Eur. J. Biochem. 269) 5199
whether MCM2 and pol II holoenzyme bind to each other
in these organisms. In this context it is important that MCM
proteins were not detected in any of the S. cerevisiae pol II
holoenzyme or mediator complexes despite extensive bio-
chemical analyses [2,8,14,56].
Functional significance of the pol II holoenzyme–MCM
interaction
The functional importance of the established interaction
between MCM proteins and pol II holoenzyme remains
enigmatic. MCM proteins are components of the DNA
replication licensing machinery [24,35]. Their association
with pol II holoenzyme could reflect some function of the
latter complex in replication. Several findings are in tune
with this idea. Recruitment of pol II holoenzyme to
origins of DNA replication in S. cerevisiae significantly
stimulates their activity [45]. In addition, transcriptional
activators, most of which interact with pol II holoenzyme,
stimulateDNAreplicationwhentetheredtoviralor
cellular origins [57–62]. We found a genetic interaction
between pol II CTD and mcm5 andalsoshowedassoci-

ation of pol II with origins of DNA replication in
S. cerevisiae (K. Yankulov, D. Kramer & R. Dziak.,
unpublished observations). Control of mammalian origins
of replication is less well understood, yet it has been
reported that mammalian pol II holoenzyme complexes
contains proteins, which function in DNA repair and
replication [3,19]. Furthermore, some mammalian origins
of DNA replication have been found within mammalian
enhancers [63,64], which presumably interact with pol II
holoenzyme.
Another possibility is that MCM proteins function in
pol II transcription. For example, stimulation of IFN-c
responsive genes is significantly decreased by mutations in
Stat1a, which also preclude the association of this protein
with MCM3/MCM5 complex [43,44]. We showed that
antibodies against MCM2 inhibit pol II transcription in
injected Xenopus oocytes. Effects on DNA replication were
not analyzed because these cells do not replicate DNA [6].
We also found some transcriptional deficiencies in mcm5
mutants in S. cerevisiae (K. Yankulov and D. Leishman,
unpublished results). Preliminary experiments also indicate
consistent inhibition of the expression of a plasmid-borne
reporter gene upon overexpression of MCM2 with muta-
tions in the holoenzyme interacting domain (data not
shown).
It seems that the observed association between MCM
proteins and pol II holoenzyme could reflect some addi-
tional roles of these two complexes in pol II transcription
and DNA replication, respectively. At present, the mech-
anism of action that signifies this association is unclear. It is

conceivable that histones, pol II holoenzyme, and ORC
could come in close proximity via contacts with MCM2 to
positively (or negatively) regulate origin function. MCM2
may also have some unknown role in mediating pol II–
histone contacts. Clearly, addressing these questions
requires an in vivo system where effects on DNA replication,
transcription, and the cell cycle can be analyzed. The
identification of mutations in MCM2, which preclude its
interaction with pol II holoenzyme, is therefore a major step
towards such a detailed analysis. MCM2(169–212) peptides
or full length MCM2 with mutations within the pol II
holoenzyme binding region can be tested for dominant
negative effects in stably transfected cells. This approach
will provide opportunities for a focused functional analysis
of the MCM–pol II holoenzyme interaction.
ACKNOWLEDGEMENTS
We thank N. Thompson and I. Todorov for gifts of antibodies;
I. Todorov for the pBS/BM28 plasmid; R. Lu for help with generation
of antibodies; R. Mosser, A. Wildeman, D. Evans, J. Bag, G. Harauz,
D. Leishman for valuable suggestions and discussion. This study was
supported by a grant from Canadian Institutes for Health Research
(CIHR no. 36371) to K. Yankulov.
Fig. 8. Characteristics of the MCM2(169–212) peptide. The relative positions of the histone H3, pol II holoenzyme, HBO1 binding sites and the
ATP homology domain in MCM2 are shown (not to scale). Similarity search and multiple sequence alignment to the approximate human pol II
holoenzyme-binding sequence were performed by
BLAST
. Amino acid residues identical to the human ones are represented by (.). Overall percentage
homology to the human peptide is shown under the name of the species. (*) indicates a potential site of phosphorylation. The solid bar above the
human sequence indicates predicted helices in the peptide. Bolded text highlights the amino acids that we substituted for in our point mutation
analyses.

5200 L. Holland et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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