From the Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021
Received for publication, October 15, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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The Mcm2-7p heterohexamer is the presumed
replicative helicase in eukaryotic cells. Each of the six subunits is
required for replication. We have purified the six Saccharomyces
cerevisiae MCM proteins as recombinant proteins in
Escherichia coli and have reconstituted the Mcm2-7p complex
from individual subunits. Study of MCM ATPase activity demonstrates
that no MCM protein hydrolyzes ATP efficiently. ATP hydrolysis requires
a combination of two MCM proteins. The fifteen possible pairwise
mixtures of MCM proteins yield only three pairs of MCM proteins that
produce ATPase activity. Study of the Mcm3/7p ATPase shows that an
essential arginine in Mcm3p is required for hydrolysis of the ATP bound
to Mcm7p. Study of the pairwise interactions between MCM proteins
connects the remaining MCM proteins to the Mcm3/7p pair. The
data predict which subunits in the ATPase pairs bind the ATP that is
hydrolyzed and indicate the arrangement of subunits in the Mcm2-7p heterohexamer.
The minichromosome maintenance proteins
(MCM)1 are components of the
prereplicative complex (preRC) that assembles on replication origins
prior to S phase (1, 2). The six proteins (Mcm2-7p) form a
heterohexamer that is thought to be the replicative helicase in
eukaryotic cells. Consistent with this idea, MCM proteins are required
for replication initiation and elongation (3, 4). In addition, they
appear to travel with the replication fork in vivo (5).
Although helicase activity with all six MCM proteins has remained
elusive, a subcomplex of Mcm4/6/7p is a helicase (6-8). Similar to
replicative helicases in other systems, Mcm2-7p appears to be ring
shaped, consistent with electron microscope studies of Mcm2-7p and
Mcm4/6/7p (9, 10). An archaeal MCM protein also has helicase activity
and is an oligomeric ring (11-14).
MCM proteins are members of the AAA+ family of proteins (15). As their
name implies (ATPases associated with a variety
of cellular activities), members of this family are usually
ATPases and are involved in many different cellular processes. The
structures of some AAA+ family members have been solved, and provide
insight into Mcm2-7p structure and function. These structures include the heteropentameric Escherichia coli A striking feature of these AAA+ machines is the location of ATP sites
at the interface of subunits. Residues from both subunits are thought
to be required for ATP hydrolysis. An arginine residue of one subunit
is required for hydrolysis of ATP bound to a neighboring subunit. A
catalytic Arg residue that reaches across a subunit interface to
promote nucleotide hydrolysis is referred to as an arginine finger,
first described in the Ras GTP-binding protein and its cognate GAP
(GTPase activating protein) (21).
These signaling proteins are important regulators of cell growth and differentiation (22). When bound to GTP, Ras is active but is inactive
when bound to GDP. Ras hydrolyzes GTP very slowly and requires an
arginine residue from GAP to promote rapid hydrolysis. Structural and
biochemical studies have suggested that the Arg finger in GAP
stabilizes the transition state thereby greatly increasing the
catalytic rate of the Ras GTPase (21).
In this study, we report the purification of Mcm2-7p as individual
subunits. We find that the Mcm2-7p heterohexamer can be reconstituted
from these individual subunits. MCM proteins have been studied alone
and in combinations for intrinsic ATPase activity. We find that no
individual MCM protein contains significant ATPase activity even though
they each have an ATP binding site. ATPase activity is produced by a
combination of at least two MCM proteins, implying that ATPase activity
requires residues from both subunits (i.e. a catalytic
arginine from one subunit and an ATP binding site in another). Although
one may anticipate six ATPase pairs, we find that only three different
pairwise combinations of MCM proteins have ATPase activity. In-depth
study of one of these combinations, Mcm3/7p, demonstrates that Mcm3p
contributes a catalytic arginine residue for hydrolysis of ATP bound to
Mcm7p. Examination of the fifteen different pairwise combinations of
MCM proteins for physical interaction, along with the ATPase data,
indicates which subunits contribute catalytic arginine residues and
which bind ATP in the other two ATPase pairs. The results also suggest a unique arrangement of MCM proteins in a heterohexameric ring.
Buffers--
Tris/sucrose (50 mM Tris-HCl pH 7.5, 10% (w/v) sucrose); Buffer A is 20 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 2 mM DTT, and 10% (v/v) glycerol.
Buffer B is 20 mM HEPES-NaOH, pH 7.5, 0.1 mM
EDTA, 2 mM DTT, and 10% (v/v, glycerol). Buffer C is 20 mM Tris, pH 7.9 and 0.5 M NaCl.
Cloning of the MCM2-7 Genes--
Each of the MCM
genes was amplified from Saccharomyces cerevisiae genomic
DNA (S288C, MAT
The MCM2 gene was amplified using primer 2D,
5'-d(CTACAATATAACATATGTCTGATAATAGCAGCCGTAGCCGTGAGGAAGATG)-3',
which inserts a NdeI site at the MCM2 start
codon, and primer 2U, 5'-(dTAGGAATGGATCCGTTTTAGTGACCCAAGG-3'), which inserts a BamHI site 4-bp downstream of the
MCM2 stop codon. The resulting 2632-bp PCR fragment was
digested with NdeI and BamHI and was inserted
into the NdeI and BamHI sites of pET11a (Novagen)
to yield pET11a-MCM2.
The MCM3 gene was amplified using primer 3D,
5'-d(GATACAAACGTCACATATGGAAGGCTCAACG)-3' which inserts an
NdeI restriction site at the MCM3 start codon,
and primer 3U, 5'-d(TTAGTAAACAGAGCTCTGACATCAGAC)-3', which
inserts a SacI restriction site 6-bp downstream of the
MCM3 stop codon. The PCR fragment was ligated into the
pCR2.1-TOPO vector (Invitrogen) using the manufacturer's instructions
to yield pMD132. The 2923-bp NdeI-SacI fragment
from pMD132, containing MCM3, was inserted into the
NdeI-SacI sites of pET21b (Novagen) to yield
pET21b-MCM3.
The MCM4 gene was amplified using the primers, 4D,
5'-d(CTCAAGAACTTCATATGTCTCAACAGTC)-3', which inserts an NdeI
restriction site at the MCM4 start codon and 4U,
5'-d(TGATTGTAGTAGATCTCATCAGACACG)-3', which inserts a
BglII site 3-bp downstream of the MCM4 stop
codon. The 2829-bp PCR fragment was ligated into pCR2.1-TOPO
(Invitrogen) to create pMD134. The 2809-bp
NdeI-BglII fragment from pMD134, containing
MCM4, was inserted into the NdeI and
BglII sites of pET11a to yield pET11a-MCM4.
The primers used to amplify MCM5 from the yeast
genome were 5D, 5'-d(TAACTGCATATGTCATTTGATAGACCG)-3', which inserts an
NdeI restriction site at the MCM5 start codon and
5U, 5'-d(GCTAAGACTTAGATCTTGTCATACACCAC)-3', which inserts a
BglII restriction site 3 bp downstream of the MCM5 stop codon. The-2535 bp PCR fragment was amplified,
digested with NdeI and BglII, and then inserted
into the NdeI and BamHI sites of pET16b (Novagen)
to yield pET16b-MCM5.
The MCM6 gene was amplified using the primers 6D,
5'-d(CTGGTTTTTTCATATGTCATCCCCTTTTCC)-3', which inserts an
NdeI site at the start codon of MCM6 and 6U,
5'-d(GAAAATCCGCAAGGATCCACTGAATTAGC)-3', which inserts a
BamHI site 8-bp downstream of the MCM6 stop
codon. The 3086-bp PCR fragment was digested with NdeI and
BamHI and inserted into the NdeI and
BamHI sites of pET16b to yield pET16b-MCM6.
The MCM7 gene was amplified using primer 7D,
5'-d(GATAGACCAGATCATGAGTGCGGCACTTC)-3', which inserts a
BspHI restriction site at the MCM7 start codon, and primer
7U, 5'-d(GTTGCTTGACGTCGCTGAGCTTTCAAGCGTC)-3', which inserts a
BlpI restriction site 5 bp downstream of the MCM7 stop codon. The resulting 2545-bp PCR fragment was inserted into pCR2.10-TOPO to yield pMD131. The 1491-bp BspHI fragment
from pMD131, encoding an N-terminal fragment of Mcm7p, was inserted into the NcoI site of pET16b to yield pMD135. A plasmid
encoding full-length Mcm7p (pET16b-MCM7) was produced by inserting the 2075-bp BamHI-BlpI fragment from pMD131 into the
BamHI-BlpI sites of pMD135.
SRF and P-loop Mutations of MCM3 and MCM7--
For the Mcm3p SRF
mutation, the Arg at residue 542 was changed to alanine so that
nucleotides 1761-1765 (numbering begins at the start codon) were
changed from 5'-d(TCTCG)-3' to 5'-d(AGCGC)-3'. The Arg of the Mcm7p SRF
motif (R593) was changed to alanine by altering nucleotides 1924-1929
from 5'-d(TCCAGA)-3' to 5'd(AGCGCA)-3', also introduces a
HaeII restriction site. The lysine at residue 415 in Mcm3p
was replaced with alanine by changing nucleotides 1373-1378 from
5'-d(AAGTCC)-3' to 5'-d(GCTAGC)-3', which also introduces a
NheI restriction site. Lysine 466 in Mcm7p was changed to
alanine by PCR. A portion of the MCM7 gene was amplified
from an internal BamHI site to a Bsu36I
restriction site using the primers 7BAM,
5'-d(CACGATGGATCCACCCTCTTC)-3', which anneals across the
BamHI site and 7KA,
5'-d(GACCAACGCCTGAGGAACCCTTACCAGTGGTATACACTCCTCGAGGTGATATTTTGCAAATGGCCTTCAGCAGTTGAGATGCGGCAACACCGGGATCACC)-3', which changes the lysine codon to alanine and anneals across the Bsu36I site. The amplified fragment was inserted into
pET16b-MCM7 digested with the same restriction enzymes. All mutant
genes were sequenced to confirm the changes and mutant proteins were
purified in the same manner as their wild type counterparts (see below).
Cell Growth and Lysis--
Expression plasmids were transformed
into BL21 DE3 Codon+ RIL cells (Strategene, E. coli B
F
For cell lysis, cells were brought to a final volume of 250-300 ml
with a final concentration of 30 mM spermidine, 1 M NaCl, and 2 mM DTT in Tris/sucrose. Cells
were lysed by two passages through a French Press at 17,000 psi, and
insoluble material was removed by centrifugation at 10,000 rpm for 40 min at 4 °C in an SLA1500 rotor. The supernatant was decanted and
referred to below as the cell lysate or FrI (Fraction I).
Mcm2p Purification--
FrI of the Mcm2p expression strain was
treated with 0.25 g/ml of ammonium sulfate. After stirring at 4 °C,
the mixture was centrifuged at 10,000 rpm for 30 min in a SLA1500
rotor. The resulting supernatant was removed, and the pellet was
resuspended in Buffer A containing 0.2 g/ml ammonium sulfate and then
re-pelleted. After repeating this step, the pellet was resuspended in
100 ml of Buffer A and dialyzed against Buffer A for ~3 h. The
protein was diluted to a conductivity equal to 60 mM NaCl
with Buffer A (final volume, 220 ml) and then applied to a 225 ml of
Fast Flow Q Sepharose column equilibrated in Buffer A. Protein was
eluted with a 2250 ml, 0-600 mM NaCl gradient in Buffer A. Fractions containing Mcm2p, as determined by Coomassie-stained gels,
were pooled (FrIII, 200 ml, 1.6 mg/ml) and then precipitated with 0.25 g/ml ammonium sulfate. After centrifugation, the pellet was resuspended
in 50 ml of Buffer B and dialyzed against Buffer B for 2.5 h. The
protein was diluted with Buffer B before being applied to a 200-ml
heparin-agarose column (BioRad) equilibrated in Buffer B. The protein
was eluted with a 1750 ml, 0-500 mM NaCl gradient in
Buffer B. Fractions containing Mcm2p were pooled (FrIV, 500 ml, 0.47 mg/ml) and precipitated with 0.25 g/ml ammonium sulfate. After
centrifugation, the pellet was resuspended in 50 ml of Buffer A and
then dialyzed against Buffer A for 2.5 h. The protein was diluted
with Buffer A to a conductivity equivalent to 100 mM NaCl.
Particulate matter was removed by centrifugation at 10,000 rpm for 10 min at 4 °C in an SS34 rotor. The supernatant was applied to a 20-ml
Mono Q column equilibrated in Buffer A. The column was then washed with
Buffer A containing 200 mM NaCl before eluting the protein
with a 400 ml, 200 mM to 600 mM NaCl gradient
in Buffer A. Fractions containing Mcm2p were pooled (FrV, 50 ml, 2.7 mg/ml) and then stored at Purification of Mcm3p--
Lysate from the Mcm3p expression
strain was fractionated by adding 0.2 g/ml ammonium sulfate and stirred
at 4 °C for 30 min, before centrifugation for 30 min at 10,000 rpm
in an SLA1500 rotor. The pellet was resuspended in 200 ml of Buffer A
containing 0.2 g/ml ammonium sulfate and then re-pelleted. The pellet
was resuspended in 200 ml of Buffer A, then dialyzed against Buffer A
for ~3 h. The preparation was diluted with Buffer A to a conductivity
equal to 100 mM NaCl (final volume, 400 ml), then applied
to a 200-ml Fast Flow Q Sepharose column equilibrated in Buffer A. The
protein was eluted with a 2 liter, 0-500 mM NaCl gradient,
in Buffer A. Peak fractions containing Mcm3p were pooled (Fr III, 130 ml, 1 mg/ml) and then dialyzed against Buffer B for 2 h. The
protein was diluted with Buffer B before it was applied to a 100-ml
heparin-agarose column equilibrated in Buffer B. The column was washed
in Buffer B, then Mcm3p was eluted with a 1000-ml, 0-500
mM NaCl gradient in Buffer B. Fractions containing Mcm3p
were pooled (FrIV, 280 ml, 0.25 mg/ml) and then 0.3 g/ml of ammonium
sulfate was added. After centrifugation, the pellet was resuspended in
25 ml of Buffer A and dialyzed against Buffer A for 2 h. The
preparation was diluted with Buffer A to a conductivity equivalent to
50 mM NaCl (final volume, 60 ml) before being applied to an
8-ml Mono Q column equilibrated in Buffer A. The column was washed, and
then Mcm3p was eluted with a 160-ml gradient of 0-500 mM
NaCl in Buffer A. Peak fractions containing Mcm3p were pooled (FrV, 40 ml, 3.3 mg/ml) and stored at Purification of Mcm4p--
FrI containing Mcm4p was treated with
0.3 g/ml ammonium sulfate. After centrifugation at 4 °C and 12,000 rpm in an SLA1500 rotor, the pellet was resuspended in Buffer A
containing 100 mM NaCl. Then, 0.25 g/ml ammonium sulfate
was added and re-pelleted. This step was repeated two more times. The
resulting pellet was resuspended in 150 ml Buffer A, then dialyzed
against Buffer A. The protein was applied to a 200-ml Fast Flow Q
Sepharose column equilibrated in Buffer A + 100 mM NaCl,
and the column was washed with the same buffer. Mcm4p was eluted with a
2-liter, 100-500 mM NaCl gradient in Buffer A. Peak
fractions containing Mcm4p were pooled (FrIII, 300 ml, 0.51 mg/ml) and
0.3 g/ml ammonium sulfate was added. After centrifugation, the pellet
was resuspended in 30 ml of Buffer B and dialyzed against Buffer B for
4 h. The protein was applied to a 70-ml heparin-agarose column
equilibrated in Buffer B with 100 mM NaCl, and then the
column was washed in Buffer B containing 100 mM NaCl.
Mcm4p was eluted from the column with a 700 ml, 100-500 mM
NaCl gradient in Buffer B. The peak fractions containing Mcm4p were
pooled (FrIV, 180 ml, 0.12 mg/ml) and precipitated with 0.3 g/ml
ammonium sulfate. After centrifugation, the pellet was resuspended in
10 ml of Buffer B and dialyzed against Buffer B for 2.5 h. The
protein was applied to a 1-ml Mono S column equilibrated in Buffer B,
and the column was washed with Buffer B + 100 mM NaCl.
Mcm4p was eluted from the column with a 20 ml, 100-500 mM
NaCl gradient in Buffer A. Peak fractions were pooled (FrV, 2.5 ml, 2.8 mg/ml) and stored at Purification of His-Mcm5p--
Lysis of induced cells harboring
pET16b-MCM5 was performed as described above, except that DTT was
omitted, the pH was adjusted to 7.9, and 5 mM imidazole was
added. After centrifugation to remove cell debris, the supernatant was
applied to a 50-ml Ni-charged chelating-Sepharose (Amersham
Biosciences) column equilibrated in Buffer C containing 5 mM imidazole. The column was washed with 600 ml of Buffer C
containing 5 mM imidazole and then 500 ml of Buffer C
containing 60 mM imidazole. Bound proteins were eluted with
a 500 ml, 60 mM to 1 M imidazole gradient in
Buffer C. Peak fractions containing His-Mcm5p were pooled (FrII, 40 ml,
0.25 mg/ml) and then dialyzed first against Buffer A containing 0.5 M NaCl, but no DTT, and then against Buffer A containing
100 mM NaCl. After dialysis, the preparation was clarified
by centrifugation for 10 min at 12,000 rpm in an SS34 rotor at 4 °C,
then applied to a 1-ml Mono Q column equilibrated in Buffer A with 100 mM NaCl. The column was washed with the same buffer then
eluted with a 20 ml, 100-500 mM NaCl gradient in Buffer A. Peak fractions were pooled (FrIII, 9 ml, 0.5 mg/ml) and stored at
Purification of His-Mcm6p--
The Mcm6p-expressing cells were
lysed as described above except that DTT was omitted, the pH was 7.9, and 5 mM imidazole was added to the cell slurry. After
centrifugation, the supernatant was applied to a 25-ml Ni-NTA Sepharose
column equilibrated in Buffer C containing 5 mM imidazole.
After washing the column with 800 ml of Buffer C containing 5 mM imidazole, followed by 1 liter of Buffer C with 60 mM imidazole, bound protein was eluted with a 250 ml,
60-500 mM imidazole gradient in Buffer C. Peak fractions containing Mcm6p were pooled (FrII, 125 ml, 0.56 mg/ml) and dialyzed against Buffer A for 2 h. The preparation was then applied to a
4-ml EAH Sepharose (Amersham Biosciences) column equilibrated in Buffer
A, and the column was washed with Buffer A containing 100 mM NaCl. The column was eluted with a 40-ml, 100-500
mM NaCl gradient in Buffer A (40 ml). Peak fractions were
pooled (FrIII, 65 ml, 0.6 mg/ml), dialyzed against Buffer A for 2 h, and then applied to an 8-ml Mono Q column equilibrated in Buffer A. The column was eluted with an 80 ml, 0.1-0.5 M NaCl
gradient. Peak fractions containing Mcm6p were pooled (FrIV, 30 ml,
0.44 mg/ml) and stored at Purification of Mcm7p--
Cell lysate of Mcm7p-expressing cells
was treated with 0.3 g/ml ammonium sulfate. After centrifugation for 30 min at 12,000 rpm in an SLA1500 rotor at 4 °C, the pellet was
resuspended in Buffer A containing 0.25 g/ml ammonium sulfate and then
centrifuged as above. This step was repeated using Buffer A containing
0.2 g/ml ammonium sulfate. The resulting pellet was resuspended in 100 ml of Buffer A and dialyzed against Buffer A for 2 h (final conductivity equal to 110 mM NaCl). The protein (FrII, 100 ml, 3.6 mg/ml) was applied to a 120-ml Fast Flow Q Sepharose column equilibrated in Buffer A, and the column was washed with the same buffer. The column was eluted with a 1.2-liter, 0-500 mM
NaCl gradient in Buffer A. Peak fractions containing Mcm7p were pooled (FrIII, 105 ml, 2.4 mg/ml) and treated with 0.3 g/ml ammonium sulfate.
After centrifugation, the pellet was resuspended in 100 ml of Buffer B,
then dialyzed overnight against Buffer B containing 100 mM
NaCl. The protein was diluted to a conductivity equal to 80 mM NaCl with Buffer B and then applied to a 170 ml Heparin agarose column equilibrated in the same buffer. The column was washed
with Buffer B and then eluted with a 1.5-liter, 0-500 mM NaCl gradient in Buffer B. Peak fractions containing Mcm7p were pooled
(FrIV, 450 ml, 0.30 mg/ml) and 0.3 g/ml ammonium sulfate was added.
After centrifugation the pellet was resuspended in 25 ml of Buffer A
and dialyzed against Buffer A for 2.5 h. The protein was diluted
with Buffer A before being applied to an 8-ml Mono Q column
equilibrated in Buffer A. Bound proteins were eluted with an 80-ml,
0-500 mM NaCl gradient in Buffer A. Peak fractions containing Mcm7p were pooled and then stored at Reconstitution of Mcm2-7p--
An equimolar amount (44 nmol) of
each MCM protein was mixed and concentrated using a Centricon-50
ultrafiltration device at 4 °C to a final volume of 2.5 ml and a
conductivity equivalent to 50 mM NaCl in Buffer A. After
incubation at 15 °C for 30 min, the sample was applied to a 1-ml
Mono Q column equilibrated in Buffer A. The column was eluted with a
20-ml, 0-500 mM NaCl gradient in Buffer A. A portion (3 mg
in 250 µl) of the peak fractions containing all six MCM proteins was
dialyzed against Buffer A with 50 mM NaCl and then was
applied to a Superose 6 gel filtration column as described below. A
portion (2 µl) of the peak fractions from the gel filtration column
was examined for ATPase activity as described below, except that the
reactions were incubated for 60 min at 30 °C. The presence of Mcm2p
and Mcm4p in the gel filtration fractions was determined by Western
blotting using standard protocols.
Gel Filtration Analysis--
Gel filtration was performed at
4 °C in Buffer A containing 100 mM NaCl using a Superose
6 gel filtration column (Amersham Biosciences). To test for
interactions between MCM proteins, the indicated amounts of proteins
were mixed, diluted to a conductivity equivalent or below 100 mM NaCl and then, if necessary, concentrated to 2 mg/ml
using a Centricon-50 ultrafiltration device (Amicon). The sample was
incubated at 15 °C for 30 min. Any debris was removed by
centrifugation for 10 min at 12,000 rpm in a table top centrifuge, and
then the sample was applied to the gel filtration column and eluted in
Buffer A containing 100 mM NaCl. After the first 5.9 ml,
fractions of 175 µl were collected at 4 °C. A portion of each fraction, as indicated, was analyzed by SDS-PAGE (6%). Densitometric analysis was performed using a laser densitometer and ImageQuant software (Molecular Dynamics).
Nucleotide Hydrolysis--
Nucleotide hydrolysis was measured by
thin layer chromatography. For analysis of individual MCM proteins and
the pairwise combinations, each reaction (15 µl) contained 1 mM [ Assembly of Mcm2-7p from Individual Subunits--
Mcm2-7p copurify
as a heterohexamer from many different eukaryotes (9, 24-27). Assembly
of Mcm2-7p may require a certain phosphorylation state, or may require
a particular order of subunit addition (i.e. like the
To determine whether these six recombinant MCM proteins could assemble
into a heterohexamer, the six individual MCM proteins were combined and
analyzed by ion exchange chromatography on a Mono Q column and then by
gel filtration. Results of the Mono Q column showed that several MCM
proteins resolved from one another, but toward the end of the gradient
all six of the proteins co-eluted, indicating that the Mcm2-7p complex
may have assembled (not shown). Some MCM proteins (Mcm2p and/or Mcm4p)
appeared in stoichiometric excess, indicating that the putative hexamer
formed but eluted along with excess single subunits. Fractions
containing the putative Mcm2-7p complex were pooled and analyzed by gel
filtration on a Superose 6 column to separate any free proteins from
the heterohexamer (Fig. 1B). All six subunits co-eluted at
the expected size of a Mcm2-7p hexamer comprised of one of each subunit
(607 kDa) suggesting that heterohexamer had formed. The complex
resolves from the excess Mcm2p/Mcm4p and elutes earlier than any of the
MCM proteins analyzed individually (compare Figs. 1 and 4A).
Furthermore, the Mcm2-7p complex elutes earlier that any of the
pairwise complexes described later (see Fig. 4B). However,
it is still possible that other subcomplexes, comprised of fewer
subunits, elute at the same position as Mcm2-7p heterohexamer. Indeed,
there appears to be some slight variation in subunit ratios in
different fractions (compare the Mcm2/4 band in fractions 30 and 32 in
Fig. 1B). Mcm2p and Mcm4p did not resolve in the SDS gel,
but the presence of both proteins in the Mcm2-7p heterohexamer was
confirmed by Western blotting (not shown). The results indicate that
assembly of the Mcm2-7p complex can proceed without a particular
subunit order, or need for phosphorylation.
It has been reported that the Mcm2-7p heterohexamer in budding yeast is
an ATPase (29), however in other systems such an activity has not been
detected (7). Fig. 1C shows that the majority of ATPase
activity co-elutes with Mcm2-7p (30 pmol/µg/min in fraction 32).
Addition of single strand DNA did not affect this activity (not shown).
A weaker, but significant ATPase activity is associated with later
fractions (fractions 38-48) that do not appear to contain all six
subunits. As we will show later in this study and has been shown in
other studies (29), there are pairwise complexes of MCM proteins that
contain ATPase activity (see Fig. 2A). Thus some of the activity
in fractions 38-48 may be the result of those smaller complexes. As
noted above, there are some slight variations in MCM protein ratios
from fraction to fraction. We cannot completely rule out the
possibility that the ATPase activity in these fractions is due to
contaminating subcomplexes comprised of fewer than six subunits or that
the hexamer dissociates into smaller subcomplexes when diluted into the
ATPase assay. Next, we examined the individual MCM proteins for ATPase
activity.
ATPase Activities of the MCM Subunits and Subcomplexes--
Study
of individual MCM proteins showed very little or no ATP hydrolysis
activity (Fig. 2A). Longer incubation times or higher concentrations of Mcm2p, 3p, 4p, 5p, or 6p (for example, 100 ng/µl for 60 min) did not yield significant ATP hydrolysis (data not shown).
However, when Mcm7p was assayed at a higher protein concentration, some
hydrolysis of ATP was observed (Fig. 2A, inset; 7 pmol/µg/min). The ATPase activity is likely intrinsic to Mcm7p,
rather than due to a contaminant, as mutation of the invariant lysine
in the Mcm7p ATP site (described in more detail below) resulted in a mutant protein, Mcm7pP
Each of the fifteen different combinations of two MCM proteins was
tested for ATPase activity. The results in Fig. 2A show that
three of the pairwise combinations result in ATPase activity. The
Mcm3/7p pair produced the most active ATPase (Fig. 2A;
~220 pmol/µg/min). As this study was performed, a report appeared
that supports this observation (29). In addition, ATPase activity is
associated with the Mcm4/7p pair (~104 pmol/µg/min). A still weaker
activity is associated with the Mcm2/6p pair (35 pmol/µg/min), consistent with a previous study (29). These assays were performed in
the absence of DNA; however the presence of M13mp18 single strand DNA
(330 ng) did not affect any of the ATPase activities documented herein
(data not shown). It is also interesting to note that the pairwise
complexes have significantly higher ATPase activity than a
heterohexamer containing all six MCM proteins (compare Fig.
1B with Fig. 2A). This trend of the Mcm2-7p
heterohexamer having less activity than complexes comprised of fewer
subunits has been observed by others (7, 29).
The results of Fig. 2A show that significant ATPase activity
requires at least two MCM proteins. This observation is consistent with
the location of ATP sites at the interfaces of AAA+ protein complexes.
Take for example the circular pentameric
Alignment of the Analysis of the Mcm3/7p ATP Site--
The hypothesis
that there is only one complete ATP site in Mcm3/7p, and that ATP
hydrolysis requires the Arg residue of the SRF motif of one subunit and
the ATP site of the other, was tested in the experiments of Fig.
3. The Arg residue of the SRF motif in
Mcm3p (R542) and in Mcm7p (Arg-593) were each changed to Ala and the
resulting mutant proteins, Mcm3pSAF and
Mcm7pSAF, were purified. The prediction is that only one
combination of wild type and mutant protein will result in loss of
ATPase activity, namely the one that participates in the ATP site at
the interface of the Mcm3/7p complex (see diagrams in Fig.
3A).
In Fig. 3A, the bipartite construction of the ATP site is
illustrated such that the subunit to the left contributes the
presumptive catalytic arginine, and the subunit to the right binds the
ATP to be hydrolyzed. This left-to-right or clockwise orientation is
that observed from the C-terminal face of the subunits in multimeric AAA+ machines (see
We first tested whether the Mcm3pSAF mutant retained ATPase
activity when mixed with wild type Mcm7p. The results in Fig.
3A show that the Mcm3pSAF mutant failed to
produce ATPase activity. Thus, one may predict that the Arg of Mcm7p
may not be needed for hydrolysis of ATP bound to Mcm3p. As the results
in Fig. 3A show, this prediction is upheld: the combination
of Mcm7pSAF and Mcm3p formed as active an ATPase as wild
type Mcm3/7p.
The loss of ATPase activity with Mcm3pSAF/7p
may be due to inability of the mutant to form a complex with Mcm7p. In
Fig. 3C, a mixture of Mcm3pSAF and Mcm7p was
analyzed by gel filtration to determine if they form a complex. The
results show that Mcm3pSAF and Mcm7p co-elute earlier than
either subunit alone, indicating that they form a complex. A similar
analysis using wild type Mcm3p and Mcm7p yields the same result.
The above results suggest that the Mcm3/7p ATPase binds ATP in Mcm7p,
not Mcm3p. If this is the case, then mutation of the Mcm3p ATP site
should not affect the Mcm3/7p ATPase (see diagram in Fig.
3B). To test this, the P-loop motifs of Mcm3p and Mcm7p were
mutated. The P-loop or Walker A motif is conserved in many nucleotide-binding proteins and binds the phosphate moiety of the
nucleotide (34, 35). The invariant lysine of the P-loop motif, Lys-415
of Mcm3p and Lys-466 of Mcm7p, were changed to Ala and the mutant
proteins, Mcm3pP
Overall, these results demonstrate that the Mcm3/7p ATPase binds ATP in
Mcm7p, and hydrolysis requires a catalytic Arg within the SRF motif of
Mcm3p. Interestingly, the ATPase activity intrinsic to Mcm7p may also
utilize an interfacial catalytic arginine since Mcm7p oligomerizes on
its own (discussed later, in Fig. 4). In further support of this conclusion, the Mcm7SAF mutant has
very little ATPase activity compared with wild type Mcm7p (Fig.
2A, inset).
Arrangement of MCM Proteins--
Use of a catalytic arginine in
Mcm3p for hydrolysis of ATP bound to Mcm7p predicts how the subunits
are arranged when viewed from the C termini (as in Fig. 3). By analogy
to
A stabile interaction between proteins is indicated when the elution
volume of one or both proteins is altered from its elution volume
alone. As a control, we first determined the elution volume of each of
the individual MCM proteins in the gel filtration column (Fig.
4A). The oligomeric state of each MCM protein can be
estimated from the elution volume. However the results are only
approximate since the migration of a protein through the gel filtration
resin is affected by both shape and size. The monomeric protein masses, predicted from gene sequences, are: Mcm2p, 99 kDa; Mcm3p, 107 kDa;
Mcm4p, 105 kDa; Mcm5p, 86 kDa; Mcm6p, 113 kDa; and Mcm7p, 95 kDa. The
results demonstrate that Mcm5p elutes at a position consistent with a
monomer, whereas Mcm2p, Mcm3p, Mcm4p, Mcm6p and Mcm7p elute at volumes
consistent with a size range of 200-300 kDa, and thus may form oligomers.
Stabile interaction between MCM proteins was tested by mixing pairs of
MCM proteins and analyzing their elution behavior. As illustrated in
the experiments of Fig. 3, an interaction between Mcm3p and Mcm7p is
observed using this technique (Fig. 4B, panel 9).
In addition, a complex between Mcm4p and Mcm7p is also detected (Fig.
4B, panel 12); Mcm4p and Mcm7p co-elute at a
position earlier than either protein alone, indicating that they form a
Mcm4/7p complex. A Mcm4/7p complex is consistent with the production of ATPase activity by this pair of proteins. Densitometric analysis reveals that the ratio of Mcm3p to Mcm7p (Fig. 4B,
panel 9), and of Mcm4p to Mcm7p (Fig. 4B,
panel 12), in the peak fractions is 1:1 and 0.9: 1, respectively.
In addition to the Mcm3/7p and Mcm4/7p complexes, interactions are also
detected between Mcm3p and Mcm5p, and between Mcm4p and Mcm6p (Fig.
4B, panels 7 and 11, respectively).
Interactions between Mcm3p and Mcm5p have been reported previously in
many different systems (7, 24, 27, 36-39). Consistent with the previous isolation of this complex (7), the Mcm3/5p complex elutes at a
volume consistent with a heterodimer containing one each of Mcm3p and
Mcm5p (predicted size, 188 kDa). Densitometric analysis indicates a
ratio of 1:1 Mcm3p to Mcm5p in the peak fraction. The Mcm4/6p complex
peaks at an elution volume consistent with a hetero-oligomer and the
ratio of Mcm4p to Mcm6p is ~1:1 in the peak fraction.
Analysis of Mcm2p and Mcm6p by gel filtration gave no conclusive
evidence of stabile complex formation (Fig. 4B, panel
4). This combination provides ATPase activity and thus is expected to form a complex. The mixture of Mcm2p and Mcm6p show that the two
proteins co-elute from the gel filtration column, suggesting that they
form a complex. Individually, Mcm2p and Mcm6p each elute as oligomers
and migrate at the same position as the Mcm2/6p mixture. Thus, it is
possible that a Mcm2/6p hetero-oligomer forms, but elutes at the same
position as the homo-oligomers. Alternatively, the interaction between
Mcm2p and Mcm6p, detected in the ATPase assays, may not be stabile to
gel filtration. Similarly ambiguous results were also obtained with the
combinations of Mcm2/3p, Mcm2/4p, Mcm3/4p, Mcm3/6p, and Mcm6/7p (Fig.
4B). In the absence of any convincing evidence of
interaction between these pairs of proteins, we conclude that they do
not interact. The protein-protein interactions that are observed,
assuming the Mcm2/6p ATPase pair also interact, along with the
documentation of an Mcm2-7p heterohexamer in many different systems (7,
9, 24-27, 29) predicts a unique arrangement of MCM proteins in a
heterohexameric ring (Fig. 5).
Reconstitution of Mcm2-7p and Arrangement of Its Subunits--
The
individual MCM proteins have been expressed in E. coli
and purified. These subunit preparations can be simply mixed together to reconstitute the Mcm2-7p heterohexamer, indicating that there is no
obligatory subunit order of addition, or need for a particular phosphorylation state. No individual MCM protein displays significant ATPase activity even though they each have an ATP site. However, certain pairs of MCM proteins have ATPase activity. Examination of one
of these ATPase pairs, Mcm3/7p, has revealed only one competent ATP
site in which Mcm7p binds ATP and Mcm3p contributes a catalytic Arg
residue. We also document which combinations of two proteins form
hetero-oligomer pairs. The protein-protein contacts, along with
information from the ATPase analysis, lead to a proposed arrangement of
MCM proteins in a simple ring (Fig. 5). This model is not predicated in
the Mcm2-7p heterohexamer having ATPase activity.
The proposed MCM ring includes the pairs of ATPases observed in Fig. 2
of this study: Mcm2/6p, Mcm3/7p, and Mcm4/7p, as well as the complexes
observed in the gel filtration analysis of Fig. 4: Mcm3/5p, Mcm3/7p,
Mcm4/6p, and Mcm4/7p. Protein-protein interaction studies
herein indicate how the other subunits connect with Mcm3/7p and these
side-by-side interactions are consistent with a ring as illustrated in
Fig. 5. The ring in Fig. 5 is viewed from the C-terminal face of the
complex. The arrangement predicts that Mcm4p of the Mcm4/7p ATPase
binds the ATP, and that Mcm7p contributes the catalytic Arg residue.
Likewise, the model predicts that the Mcm2/6p ATPase binds ATP in
Mcm2p, and that Mcm6p contributes the catalytic Arg residue. For each
of the six MCM proteins, mutation of the P-loop in the ATP binding site
results in loss of cell viability in S. cerevisiae (29).
Thus, each subunit is thought to interact with ATP. Whether all six
subunits also hydrolyze ATP is unknown. Indeed, we cannot fully
determine whether any of the ATP sites are active in the Mcm2-7p
heterohexamer. Our ability to detect only three ATPase pairs should not
be taken to imply that the other three subunits are not catalytic
(explained in more detail later).
The interactions observed in this study are also consistent with
studies of MCMs from diverse organisms which have revealed not only the
Mcm3/5p and Mcm3/7p complexes, but other complexes such as Mcm4/6/7p
and Mcm2/4/6/7p (6, 7, 27, 29, 36-41), all of which can be assembled
in vitro from these individual
subunits.2 An interaction
between Mcm2p and Mcm4p has been reported for mammalian MCMs (42).
However, this interaction was in the presence of Mcm6p and thus is also
consistent with the model in Fig. 5. There may be additional
interactions between MCM proteins that are not easily observed by gel
filtration analysis or implied by ATPase analyses. Interestingly, we
did not observe an interaction between Mcm2p and Mcm5p. Perhaps, the
Mcm2-7p ring has a gap between these subunits. Alternatively, Mcm2p and
Mcm5p may interact, but an interaction was not detected under the
conditions used here.
In the arrangement proposed in Fig. 5, the Mcm4/6/7p subunits form one
half of the hexameric ring and the Mcm2/3/5p subunits form the other
half. Previous studies in other systems have shown that the Mcm4/6/7p
complex unwinds DNA, and Mcm2p and Mcm3/5p complex interferes with this
unwinding activity (6, 7). In those studies, the Mcm4/6/7p complex was
shown to be a heterohexamer, probably a dimer of the Mcm4/6/7p
heterotrimer. Electron microscope studies have revealed that the human
Mcm4/6/7p complex is ring-shaped (10). Perhaps Mcm2p, 3p and 5p replace
one of the Mcm4/6/7p trimers when Mcm2-7p is formed.
Why Bind ATP at an Interface?--
The arrangement of ATP sites at
the interface of subunits has implications for protein function. The
placement of residues important for ATP binding and hydrolysis at one
site in two different subunits provides a basis for communication
between the subunits. For example, the ability of one subunit to
perform a certain function or activity may be dependent on the
nucleotide bound state of the neighboring subunit, which in turn could
be sensed or communicated through positioning the catalytic arginine in
or out of the ATP site. Specifically, ATP binding by one subunit may
cause allosteric conformational changes in that subunit that may take
the catalytic arginine located on its other side and place it in the
proper orientation for catalysis of ATP bound to the neighboring
subunit, triggering ATP hydrolysis.
Why Not Six ATPase Pairs?--
ATPase analysis of pairwise
combinations of MCM proteins revealed only three different ATPase
pairs. One might expect six different ATP sites in the heterohexamer,
one at each subunit interface, and therefore six ATPase pairs. Indeed,
mutation of the P-loop of any one of the six MCM proteins inactivates
Mcm2-7p (29). One possible explanation for detecting only three ATPase pairs may be that the three "inactive" pairs, Mcm2/5p, Mcm3/5p, and
Mcm4/6p, require something extra to yield an active ATPase. For
example, the arginine finger may not be positioned properly within the
subunit pairs. Proper positioning may require interaction with other
MCM proteins within Mcm2-7p. Alternatively, other factors may also be
required. For example, DNA and/or other protein components of a
replication fork may be required for all the sites to be active, and
requiring their presence may ensure that Mcm2-7p is not active until it
is properly arranged within a larger machinery.
It is also possible that not all of the ATP sites within Mcm2-7p are
catalytic. A model of Mcm2-7p has been proposed in which three MCM
proteins are non-catalytic, regulatory subunits (29). In this model
there are two trimeric rings, one stacked upon the other. One ring
contains the regulatory subunits and the other ring contains the
catalytic subunits. The ATP sites are proposed to be at the interfaces
between the regulatory and catalytic trimeric rings. This model nicely
incorporates the observations that Mcm4/6/7p (the putative catalytic
ring) is a helicase and Mcm2, 3, and 5p (the proposed regulatory ring)
inhibits the helicase. However, the structures of AAA+ multi-protein
machines show that ATP sites are formed at subunit interfaces that are
"in plane" with one another, and thus the structural data are not
consistent with a model in which ATP sites are formed at the stacking
interface between two different protein rings.
The fact that only some ATP sites are utilized in a protein ring has
been described for other helicases. For example, the T7 phage
replicative helicase, gp4, is a ring shaped hexamer (43) that binds
nucleotide with negative cooperativity (44). Presumably, some
unoccupied sites are important to overall function of the hexamer. This
negative cooperativity, along with other biochemical studies, led to
the proposal of a mechanism similar to that of F1 ATPase
(45). This idea is largely upheld by structural studies of T7 gp4 (46).
The structure suggests that the negative cooperativity is due to
conformational changes induced in the ring by binding of nucleotide. A
negative cooperative mechanism is certainly possible with Mcm2-7p, but
since Mcm2-7p is a heterohexamer, it could simply contain some subunits
that bind ATP, but do not hydrolyze it. Alternatively, all six MCM
subunits may be catalytic, as indicated by the presence of an SRF motif
in each subunit.
The exact role of the MCM heterohexamer in chromosomal replication is
not understood. Mcm2-7p is the best candidate for the replicative
helicase in eukaryotic cells. Consistent with a role at the replication
fork, the MCM proteins associate with the origin before initiation of
DNA replication (1, 2), and examination of Mcm4p and Mcm7p indicate
that they travel with the replication fork (5). Only a subcomplex of
Mcm4/6/7p has helicase activity in vitro; no helicase
activity has been detected with Mcm2-7p (7). However, all six of the
MCM proteins are required for ongoing replication in S. cerevisiae (3). One possible explanation for these seemingly
conflicting observations is that Mcm2-7p heterohexamer requires the
context of a replication fork for unwinding activity. For example, the
unwinding activity of the E. coli replicative helicase is
much greater when coupled to the replicative polymerase (47). A clear
answer to the question of MCM function in DNA replication awaits the
reconstitution of a eukaryotic replication fork in
vitro.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
complex clamp
loader (16), the homohexameric membrane trafficking proteins,
N-ethylmaleimide sensitive factor (NSF) and p97 (17-19),
and the homohexameric branch migration protein, RuvB (20).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SUC2 mal mel gal2 CUP1 flo1 flo8-1; from ATCC)
using either Vent polymerase (New England Biolabs) or Elongase enzyme
mix (Invitrogen). Yeast genomic DNA was obtained as described
previously (23). Amplified genes were ligated, as described below, into
various pET plasmids so that expression of the MCM genes is
under control of the T7 RNA polymerase promoter and inducible by IPTG.
All genes were sequenced to ensure that there were no mutations
introduced by PCR.
ompT hsdS (rB
mB
) dcm+
Tetr gal (
DE3) endA Hte
(argU ileY leuW Camr)). Fresh transformants were
grown in 24 liters of LB containing 100 µg of ampicillin and 25 µg
of chloramphenicol per ml to a density of OD600 = 0.5-0.8.
Cells were then chilled to 15 °C on ice, and then IPTG was added to
1 mM. Cells were incubated another 20 h with shaking
at 15 °C, then pelleted and resuspended in Tris/sucrose (total
volume, ~200 ml).
80 °C.
80 °C.
80 °C.
80 °C.
80 °C.
80 °C.
-32P]ATP (8-30 µCi/ml; PerkinElmer
Life Sciences), 20 mM Tris acetate, pH 7.5, 10 mM magnesium acetate, and 2 mM DTT as well as
0.5 µM of each MCM protein. Reactions were incubated for
30 min at 30 °C and then quenched by addition of 15 µl of 50 mM EDTA, pH 8.0. For the Mcm3/7p mutant study, 18 µM of Mcm3p and 0.5 µM of Mcm7p, mutant or
wild type, was used, and the reactions were incubated for 20 min at
30 °C. A portion (1 µl) of each reaction was spotted onto a
polyethyleneimine cellulose thin layer chromatography sheet (EM
Science) and then developed in 0.6 M potassium phosphate pH 3.4 for 35 min. The amount of ADP produced was quantitated using a
PhosphorImager and ImageQuant software (Molecular Dynamics). After
determining the volume of the ADP spot and the ATP spot, the amount of
ADP produced (in µM) was determined by: ((volume ADP)/(volume ADP + volume ATP)) × 1000 µM.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
'
clamp loader, Ref. 28). To test this we
constructed E. coli expression plasmids for each MCM protein
and purified the individual MCM proteins as described under
"Experimental Procedures." Mcm5p and Mcm6p had low yields and
therefore ten histidine residues were placed on the N termini of these
proteins for purification by nickel affinity chromatography. Yields
were 10-200 mg of pure protein from 24 liters of cell culture. The
final preparation of each protein is shown in Fig.
1A.
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Fig. 1.
Purification and reconstitution of Mcm2-7p.
A, Coomassie Blue-stained SDS-polyacrylamide gel (6%) of
2.5 µg of each MCM protein. Mcm2p (99 kDa) and Mcm6p (113 kDa) both
migrate slightly larger than their predicted sizes. B,
Mcm2-7p-containing fractions from a Mono Q column were pooled and
analyzed by gel filtration. The fraction numbers are above
the gel, and the migration of size standards is indicated at the
bottom. Each MCM protein is identified on the
left of the gel. C, ATPase assays of the gel
filtration column fractions. The assays were incubated for 60 min.
Fraction numbers correspond to the fractions in B.
View larger version (50K):
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Fig. 2.
ATPase analysis of MCM proteins.
A, each MCM protein, individually or in combinations of two,
was analyzed for ATPase activity. The inset shows ATP
hydrolysis by Mcm7p, wild type or mutant, under more sensitive
conditions; other single subunits gave no signal. B,
schematic of the E. coli complex clamp loader, based on
the structure looking down the C-terminal face (left
diagram) or from the side (right diagram) (16). The
arginine fingers are indicated by SRC, and the ATP binding
sites by P-loop. C, alignment of Mcm2-7p and the
(DnaX) and
' (HolB) subunits of
complex. The Mcm2-7p
proteins were aligned using ClustalX (48). Only the sequences within
the boxes defined in Ref. 15 are shown. In addition, the
corresponding sequences in the
and
' subunits are shown
below the consensus line and were positioned
according to the alignment in Ref. 15. In the consensus line, positions
that are fully identical are marked by a star; the
colon marks positions that are highly conserved residues,
and the dot marks those that are less conserved. Identical
amino acids at a given position are boxed in
black, similar amino acids at a given position are
gray.
loop that lacked significant ATPase
activity (Fig. 2A, inset). We next tested pairwise
combinations of MCM proteins for ATPase activity.
3
' clamp loader. Only the
subunits bind ATP and thus the pentamer contains three ATP sites (16, 30). The three ATP sites are located at the
'/
1,
1/
2 and
2/
3 interfaces (Fig. 2B).
Residues from both subunits are important for ATP hydrolysis: One
subunit binds ATP and the neighboring subunit contains an arginine
residue, which is thought to be important for catalysis. The putative
catalytic arginine is embedded in an "SRC" motif in both
and
', and this motif is conserved in clamp loader subunits from
bacteria, T4 phage, eukaryotes, and Archaea (31-33).
and
' sequences with MCM proteins
of budding yeast, shows that each MCM subunit contains an "SRF"
motif in the same position as the SRC motif of the clamp loader
subunits (Fig. 2C), suggesting that this arginine may be
catalytic. This motif is also conserved in MCM proteins from other
species (not shown). The fact that individual MCM proteins have very
little or no ATPase activity, and that ATPase activity is detected when two subunits are mixed, is consistent with ATP sites that require a
catalytic arginine from a second subunit.
View larger version (36K):
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Fig. 3.
Mutation of the Mcm3p SRF motif eliminates
Mcm3/7p ATPase activity. The effect of changing the conserved Arg
in the SRF motif and the conserved Lys of the P-loop motif of either
Mcm3p or Mcm7p on the Mcm3/7p ATPase was determined. A,
mutation of the SRF motifs in Mcm3p and Mcm7p is expected to correspond
to one of the two predictions shown. The results, shown as a percentage
of wild type Mcm3/7p activity, indicate that the prediction on the
right is the correct model. B, the results of mutating the
P-loop motifs in Mcm3p and Mcm7p lead to the prediction shown here. The
prediction is fulfilled by the results, expressed as a percentage of
wild type Mcm3/7p activity. C, mutant and wild type Mcm3p
and Mcm7p, together or separately, were analyzed by gel filtration.
Fraction numbers for each analysis are shown above the top
gel, and the elution positions of molecular size standards are shown
below the bottom gel. Positions of the Mcm3p and 7p are
shown to the right of each gel.
complex in Fig. 2B). Two subunit
arrangements for the Mcm3/7p complex are possible. The arrangement to
the left in Fig. 3A predicts that only mutation
of the arginine in Mcm7p will decrease ATPase activity; the
corresponding mutation in Mcm3p should have no effect. Conversely, the
arrangement to the right predicts that only mutation of the arginine in
Mcm3p will have an adverse effect on the Mcm3/7p ATPase.
loop and Mcm7pP
loop, were
purified and examined for ATPase activity in combination with the
appropriate wild type protein. The results, in Fig. 3B, confirm the prediction that mutation of the Mcm7p P-loop produces an
inactive ATPase with wild type Mcm3p. Also, as predicted, mutation of
the Mcm3p P-loop has no effect on the Mcm3/7p ATPase. This result is
consistent with a coincident study on P-loop mutants of Mcm3p and Mcm7p
(29). Gel filtration analysis of Mcm3p and Mcm7pP
loop
show that the two proteins interact, and thus the inability to form a
complex is not the cause for the loss of ATPase with
Mcm3p/7pP
loop (Fig. 3C).
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Fig. 4.
Physical interactions between MCM
proteins. Interactions among the MCM proteins were examined by gel
filtration analysis as described under "Experimental Procedures."
Analysis of the individual MCM proteins is shown in the first group of
six. Column fraction numbers are shown above the top gel.
The second group comprises the analysis of each of the fifteen pairs of
MCM proteins. The elution positions of size standards are shown
below the last gel. Each MCM protein is identified to the
right of each SDS gel.
3
', Mcm7p is immediately clockwise of Mcm3p
when viewed from the C-terminal face of these proteins, just as
1 is clockwise of
' (Fig. 2B). Next we
examined the fifteen different pairwise combinations of MCM proteins
for interaction by gel filtration to fill in the other positions around
the MCM ring.
View larger version (38K):
[in a new window]
Fig. 5.
Model of the Mcm2-7p heterohexamer. A
model based on the interactions observed in the gel filtration analysis
and ATPases analyses in this study as well as previous studies is
shown. The orientation of the subunits around the ring is based on the
view from the C-terminal face of multimeric AAA+ proteins, such as complex, in which the catalytic arginine in one subunit is clockwise of
the ATP site in the other. The orientation of all subunits is anchored
in the orientation of the Mcm3/7p ATPase pair established in Fig. 3.
SRF represents the catalytic arginine motif and
P-loop represents the ATP binding site.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank David Jeruzalmi for helpful discussions. The assistance of Jeff Finkelstein and Howard Chi is also greatly appreciated.
![]() |
FOOTNOTES |
---|
* This work was supported by the Howard Hughes Medical Institute and National Institutes of Health Grant 38839.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.
To whom correspondence should be addressed. Tel.: 212-327-7251;
Fax: 212-327-7253; E-mail: odonnel@rockvax.rockefeller.edu.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M210511200
2 M. J. Davey and M. O'Donnell, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
MCM, minichromosome
maintenance;
pre-RC, pre-replicative complex;
AAA+, ATPases associated
with a variety of cellular activities;
NSF, N-ethylmaleimide-sensitive fusion protein;
GAP, GTPase-activating protein;
DTT, dithiothreitol;
IPTG, isopropyl-1-thio--D-galactopyranoside.
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