From the Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853-2703
Received for publication, January 21, 2003 , and in revised form, April 30, 2003.
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ABSTRACT |
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INTRODUCTION |
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Like Mcm1, the MCM27 family of proteins is evolutionarily conserved from yeast to human (2). Several studies suggest that these six proteins form the precursor complex of the hexameric helicase (2224), which plays an essential role in the early steps of replication initiation (2527). The Mcm2Mcm7 proteins are assembled as components of the pre-replication chromatin during early G1 phase. In coordination with this cell cycle-regulated program, all six MCM genes are expressed in a cell cycle-specific manner, with their transcript levels reaching a peak at M/G1 or G1 phase (13, 2830). However, the notion that the periodic synthesis of these proteins might limit DNA replication to once per cell cycle conflicts with their extreme abundance and constitutive presence in proliferating cells (3134). A possible explanation for these contradictory properties is that the MCM27 proteins may perform other functions that involve separate pools of these proteins (2). This hypothesis is consistent with the observation that individual members of the MCM27 family may undergo various forms of post-translational modifications including phosphorylation (33, 35), polyubiquitination (36), and acetylation (37). Moreover, genome wide location studies indicate that individual members of the MCM27 family are localized not only at replication origins but also at nonorigin regions and that these proteins may not act in unity (38).
The possibility that Mcm1 may play an indirect role in DNA replication by regulating the expression of DNA replication genes has been investigated (18, 29, 39). In this study, we confirm that Mcm1 is involved in the transcriptional regulation of MCM7. However, we found that a cofactor of Mcm1 in this regulation is Mcm7, suggesting that Mcm7 may play a dual role in DNA replication by acting as part of the MCM helicase as well as temporally regulating its own expression.
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MATERIALS AND METHODS |
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Yeast strains used are: YC1-1 ( mcm1-1 ade2 ade3 leu2 ura3
his4), DBY2029 (
ade2-1 lys2-801 leu2-3, 112, ura3-52
mcm7-1), DBY2055 (a ura3-52 his4-619), BY4743
(a
his3/his3 ura3/ura3
leu2/leu2), K7428 (a ura3
cdc20::LEU2 his3 GAL-CDC20::TRP1), mutant
23342 (BY4743 MCM7/mcm7::Kan) (Research Genetics),
and EGY40 (a ura3 trp1 leu2 his3).
RNA MethodsRNA was purified using the yeast heat-freeze protocol (40). 10 µg of total RNA was used for Northern blots (41). Radioactive probes were produced using the STRIP-EZTM kit (Ambion, Austin, TX). Quantitation of radioactive signals was performed on a Molecular Dynamics Storm 840 PhosphorImager, and quantitative analysis was performed using ImageQuant software.
Protein PurificationMcm1-His6 was purified from 500 ml of yeast culture induced for 5 h with 2% galactose. After 5 h of induction, cells were pelleted and Mcm1-His6 was purified using the Y-PER His6 purification kit according to the protocol from the manufacturer (Pierce). Peak fractions of Mcm1-His6 were pooled and dialyzed against 2 liters of minimal Mcm1 band shift buffer (20 mM HEPES-KOH, pH 7.5, 1 mM dithiothreitol, 10% glycerol, 60 mM NaCl, 7 mM MgCl2, 1 mM EDTA).
GST fusion proteins were expressed in Escherichia coli strain
BL21(DE3) pLysS (F' ampT hsdSB
(r8,
M8) dcm gal (DE3) plysS
CmR) using pMCM1-GST or pMCM7-GST.
Isopropyl-1-thio--D-galactopyranoside-induced cells were
pelleted and re-suspended in ice-cold phosphate-buffered saline containing 1
mM phenylmethylsulfonyl fluoride. Cells were lysed by sonication
using the Branson 250 sonicator. Clarified extracts were incubated with
glutathione-S-Sepharose for 23 h at 4 °C, and the mixture
was poured in disposable columns. Columns were washed three times with 10 bed
volumes of phosphate-buffered saline before elution with reduced glutathione
at a concentration of 2 mM. Eluted samples were pooled and dialyzed
overnight against buffer 1 (20 mM HEPES, pH 7.5, 1 mM
CaCl2, 1 mM dithiothreitol, 50 µM zinc
acetate, 10% glycerol, 60 mM NaCl, 7 mM
MgCl2, 1 mM EDTA). Concentrations of purified proteins
are calibrated against known amounts of BSA in Coomassie-stained
polyacrylamide gels.
Band Shift ReactionsPurified proteins were mixed with radioactively labeled DNA in buffer 1 (20 mM HEPES, pH 7.5, 1 mM CaCl2, 1 mM dithiothreitol, 50 µM zinc acetate, 10% glycerol, 60 mM NaCl, 0.1 mg/ml BSA, 7 mM MgCl2, 1 mM EDTA) in 1530-µl volumes. 500 ng of poly(dI-dC) was added to each reaction. The binding reactions were incubated at room temperature for 20 min before electrophoresis in 56% 1x TBE, 5% glycerol polyacrylamide gels. Gels were dried onto 3-mm paper and either exposed to film or exposed to a PhosphorImager screen.
DNase I FootprintingDNase 1 footprinting was performed as described for the band shift reactions. Purified proteins and DNA were mixed in buffer 1 in a 0.2-ml volume for 20 min. A pre-calibrated amount of DNase I (Sigma) was added in a volume of 0.01 ml for 1 min at room temperature. The reaction was stopped by precipitation in a mixture of ethanol, 0.3 M sodium acetate, and 1 µg of linear acrylamide. Precipitate was washed with 70% ethanol, dried, and re-suspended in formamide buffer before loading onto a 6 M urea, 6% polyacrylamide sequencing gel.
Formaldehyde Cross-linking ImmunoprecipitationFormaldehyde cross-linking was performed as described by Hecht et al. (42) with minor modifications. Strains were grown in complete medium in 10 mM phosphate buffer at pH 7.5, to A600 of 0.61.0. Between 15 and 50 ml of cells were cross-linked and then immunoprecipitated with 1.02.5 µl of specific antibodies overnight at 4 °C on a shaker. All washing steps were performed in Bio-Rad microspin columns. The collected immune complexes were washed two times in lysis buffer (50 mM HEPES/KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), two times in lysis buffer with 0.5 M NaCl, two times in the final wash buffer (10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 0.75% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA), and two times in TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Protein-DNA cross-links were reversed overnight at 65 °C. DNA samples were purified by the addition of five volumes of PB buffer and then passed through Qiagen PCR purification columns (Qiagen, Valencia, CA). Purified DNA was eluted with 40 µl of TE.
PCR AmplificationPCR was carried out using the following
conditions: 2 times (94 °C for 2.0 min, 96 °C for 1.0 min, 54 °C
for 4.0 min), 24 times (94 °C for 1.0 min, 54 °C for 1.5 min), and 1
time (70 °C for 5.0 min). 5 µCi of [-32P]dATP was
added to each PCR set up as a multiplex reaction. Each PCR contains the
ATP11 primers (negative control) in addition to primer pairs for the
MCM7, MCM5, CDC6 promoters or ARS305, respectively. The primer pair
for ATP11 is directed at the coding region of the gene. The sizes of
the PCR products for MCM7, MCM5, CDC6, ARS305, and ATP11 are
387, 226, 261, 280, and 412 bp, respectively. Quantitation of radioactive
signals was performed on a Molecular Dynamics Storm 840 PhosphorImager, and
quantitative analysis was performed using ImageQuant software. Unlike
real-time PCR, time points with high signals may be underrepresented in
magnitude because these values may have been generated beyond the exponential
phase of the PCR reaction.
Other MethodsPlasmid stability assay is described by Lei
et al. (32). Flow
cytometric analysis was performed as described elsewhere
(43). -Galactosidase
assays were performed as described elsewhere
(44).
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RESULTS |
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To explore these possibilities, we compared the transcript levels of
MCM7 in the mcm1-1 mutant and wild type strains
(Fig. 1C, a). In
parallel, we also compared the transcript level of MCM7 in the
mcm7-1 strain (Fig. 1C,
b). We found that the MCM7 transcripts are
reproducibly reduced by approximately half in the mcm1-1 strain but
increased by 24-fold in the mcm7-1 mutant (see also
Fig. 4, B and
C). The effect of mcm7-1 on MCM7
expression is unexpected, suggesting that Mcm7 may also be involved in the
regulation of its own transcription. We also investigated the functional
relationship between Mcm1 and Mcm7 in the mcm1-1 mcm7-1 double
mutant. We found that mcm1-1 partially suppresses the growth defect
of mcm7-1 at 34.5 °C, supporting the notion that these two
mutations may exert compensatory effects
(Fig. 1D).
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To investigate whether small changes in the transcript level of
MCM7 would have a measurable effect on DNA replication, the stability
of minichromosomes in diploids containing either one or two copies of
MCM7 was examined. Northern blot analysis confirmed that there is a
2-fold difference in the transcript level corresponding to the number of
copies of MCM7 in these two strains (data not shown). The stability
of three different minichromosomes, each containing a different replication
origin, was analyzed. Minichromosomes containing ARS1 and ARS120, but not
ARS121, showed an increased loss rate in the diploid containing a single copy
of MCM7, compared with the diploid containing two copies of
MCM7 (Fig.
1E). This haploinsufficiency of MCM7 in the
maintenance of minichromosomes containing selective ARSs is similar to that
observed in a heterozygous MCM2/ diploid
(32).
Although the MCM2MCM7 genes are transcribed periodically,
reaching a peak during M/G1 phase
(13,
28), their protein products
are present constitutively at a constant level throughout the cell cycle
(33). To investigate whether
reduced MCM7 transcript level or gene dose has an effect on the Mcm7p
level in the mcm1-1 mutant and the MCM7/mcm7
diploid, Mcm7 protein levels in mutant and wild-type extracts were compared by
Western blotting. There was no measurable difference in the Mcm7p level
between the mcm1-1 mutant and parent strain
(Fig. 1F). Similarly,
no measurable difference in the Mcm7p level was observed in the
MCM7/mcm7
and the MCM7/MCM7 diploid
(Fig. 1G). These
results suggest that small changes in the MCM7 transcript level and
therefore the nascent Mcm7p pool may produce significant effects on the
stability of plasmids carrying specific ARSs. However, the bulk of the
cellular Mcm7 protein whose accumulation is independent of gene dose or
periodic expression has little effect on plasmid stability.
Mcm7 Stimulates Mcm1 Binding to the MCM7 Promoter Although
Mcm7 is not known to be a transcription factor or to bind DNA, Mcm1 has been
shown to bind sequences upstream of the MCM7 promoter
(18). To investigate whether
Mcm7 binds either alone or together with Mcm1, a 60-bp DNA fragment upstream
of the MCM7 promoter was used as the binding substrate in an
electrophoretic mobility shift assay. Mcm1 tagged either with His6
(Fig. 2A, a) or with
GST (b, lane 2), and Mcm7-GST proteins
(Fig. 2A, b, lane
1) were purified to greater than 90% homogeneity. Full-length Mcm1
migrates anomalously with an apparent molecular mass of 42 kDa
(3). The concentrations of
purified proteins were calibrated against BSA in Coomassie-stained
polyacrylamide gels. We found that Mcm1-GST and Mcm1-His6 can be
used interchangeably to produce similar results.
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Mcm7 did not bind MCM7 promoter DNA (Fig. 2B, lane 2). However, Mcm1-His6 binds the DNA fragment to form multiple Mcm1-DNA complexes (Fig. 2, B (lane 1) and C (lane 6)) with the predominant species being the highest molecular mass complex and presumably the most stable complex. These multiple species of Mcm1-DNA complexes may result from the oligomerization of Mcm1 or the binding of Mcm1 to multiple sites at the MCM7 promoter or both. To investigate the effect of Mcm7 on the binding of Mcm1 to the MCM7 promoter, Mcm7 was titrated against 6 pmol of Mcm1 (Fig. 2C, lanes 15). At 0.4 pmol of Mcm7, a molar ratio of 1:15 for Mcm7:Mcm1, a higher molecular mass protein DNA complex is detected (lane 5, arrowhead). Increasing amounts of Mcm7 yielded increasing amounts of all of the Mcm1-DNA complexes but the highest molecular mass species in particular (lanes 14).
To investigate whether this protein-DNA complex results from a specific interaction among Mcm1, Mcm7, and MCM7 promoter DNA, we carried out the binding reaction in the presence of a 50-fold excess of cold competitor DNA (Fig. 2D). Binding of Mcm1 to the MCM7 promoter DNA (lane 1) is unaffected by nonspecific competitors (lane 2) but completely competed out by specific competitor DNA (lane 3). Similarly, enhanced Mcm1 binding to the MCM7 promoter DNA in the presence of Mcm7 (lane 4) is unaffected by nonspecific competitors (lane 5) but completely competed out by specific competitor DNA (lane 6). These results suggest that the interaction between Mcm1 and Mcm7 is dependent on a specific sequence on the MCM7 promoter DNA. However, the mobility of the protein-DNA complex is indifferent to the presence of Mcm7, suggesting that Mcm7 may not associate with the Mcm1-DNA complex. We are unable to detect Mcm7 in the protein-DNA complex by mobility shift using Mcm7-specific antibodies (data not shown).
To investigate whether the stimulatory effect of Mcm7 is specific for Mcm1, we examined the effect of Mcm7 on the binding of another transcription factor AP2 to its recognition sequence. AP2 binds specifically to the AP2 binding sequence (Fig. 2E, lane 2). Addition of 8 pmol of Mcm7 to the AP2-DNA complex had no stimulatory effect on the binding of AP2 to AP2 DNA. We also examined the effect of BSA on Mcm1 DNA binding. Under the condition in which neither Mcm1 (Fig. 2F, lane 3) nor Mcm7 (lane 2) alone shows significant binding to the MCM7 promoter, addition of 25 pmol of Mcm7 to Mcm1 stimulates Mcm1 binding (lane 4) but addition of 25 pmol of BSA to Mcm1 does not (lane 5). On prolonged exposure, BSA appears to promote some Mcm1 binding to produce a smear (lane 5) rather than specific bands (lane 4). These results together indicate that the stimulatory effect of Mcm7 for Mcm1 is specific.
To investigate the nature of the interactions between Mcm1 and Mcm7 and the MCM7 promoter, DNase 1 footprinting analysis was carried out to identify the region protected by Mcm1 alone, Mcm7 alone, or Mcm1 and Mcm7 together at saturating concentrations. Mcm1 alone shows a footprint restricted to the two 16-bp palindromic ECBs of the MCM7 promoter that had been previously identified (Fig. 2G, a, lane 1, bar to the left) (18). Although Mcm7 alone does not bind MCM7 DNA in a mobility shift assay (Fig. 2F), at a saturating concentration, Mcm7 protects the AT-rich region between the two MCEs (Fig. 2G, a, lanes 2 and 3, bar to the right). When both Mcm1 and Mcm7 were added, the footprint corresponds to the combined footprints of Mcm1 and Mcm7 (a, lanes 4 and 5, bar to the right). Mcm7 appears to augment the Mcm1 footprint without extending the size of the footprint. The corresponding nucleotide sequences of these footprints are shown in Fig. 2G, b.
Electrophoretic mobility shift assay and DNase 1 footprints of Mcm1 and Mcm7 suggest that, although Mcm7 facilitates the binding of Mcm1 to DNA, Mcm7 may not be part of the stable complex. Mcm7 may facilitate Mcm1 binding by transient interactions with the DNA. Alternatively, Mcm7 may bind to the Mcm1-DNA complex but cannot survive electrophoresis. To identify the Mcm7 interacting site, mutations were introduced in the intervening sequence of the two MCEs. These mutations had no effect on either Mcm1 binding or Mcm7-stimulated Mcm1 binding (data not shown). In contrast, mutations within the MCEs either reduced or abolished Mcm1 binding (data not shown). In all, we did not identify mutations that affected Mcm7 stimulation but not Mcm1 binding. We have also investigated possible physical interactions between Mcm1 and Mcm7 in the absence of DNA. Two-hybrid analysis and co-immunoprecipitation experiments failed to detect interaction between these two proteins (data not shown).
The MCM7 Promoter Region Is Not Associated with a Replication OriginMcm7 is a subunit of the hexameric complex that is associated with replication origins. To investigate whether the association of Mcm7 with its own promoter may be the result of a fortuitous positioning of a replication origin in the promoter region of MCM7, we tested the promoter region of MCM7 for replication origin activity. The 1.6-kb intergenic region between MCM7 and its 5' upstream neighbor DER1 was inserted into YIp56, a non-replicating vector, and the resulting plasmid was tested for autonomous replication in a high frequency transformation assay (Fig. 3A). YIp56-MCM7-P DNA yielded only abortive transformants that do not survive restreaking, whereas YIp56-ARS1 yielded true transformants at a high frequency (>1000 transformants/µg DNA). This result suggests that the intergenic region between MCM7 and DER1 does not contain a replication origin.
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Mcm1 and Mcm7 Bind a Conserved Sequence in the Promoters of Early Cell Cycle GenesIf Mcm7 stimulates the binding of Mcm1 to the ECBs of the MCM7 promoter, it may also stimulate the binding of Mcm1 to promoters of other ECB genes such as SWI4, CDC6, and MCM5 (Fig. 3B) (18). The effect of Mcm7 on the binding of Mcm1 to these promoter sequences was investigated. Oligonucleotides 60 bp in length that include ECBs upstream of the promoters of MCM7, CDC6, MCM5, and SWI4 were synthesized and tested for binding with Mcm1 alone or with Mcm1 and Mcm7 together (Fig. 3C). The conditions used for the gel mobility shift assays in Fig. 3C differ from those shown in Fig. 2 (BD) in two ways. First, Mcm1-GST instead of Mcm1-His6 was used. Because GST is known to dimerize (45), it may stabilize Mcm1-GST and Mcm7-GST complexes that are otherwise unstable. Second, saturating amounts of Mcm7-GST were used to show that Mcm7-GST alone does not form a stable complex with ECB promoters (lanes 4). We found that Mcm1-GST binds MCM7 promoter DNA to yield distinct and well separated bands on PAGE (Fig. 3C, lanes 1). Mcm1-GST binding is enhanced by Mcm7-GST (lanes 3) producing well defined supershifted species with all four ECB promoters. Adding GST to Mcm1-GST did not enhance Mcm1-GST binding (lanes 2). Mcm7-GST alone does not bind ECB promoter DNA, although some binding of Mcm7 to the CDC6 promoter is detectable (lanes 4). These results suggest that, under these special conditions, Mcm1 and Mcm7 bind promoters of early cell cycle genes containing one or more ECBs.
Mcm1 and Mcm7 Regulate the Expression of Early Cell Cycle Genes in VivoMcm1 has been shown to bind constitutively to ECB promoters throughout the cell cycle (data not shown) (18). To establish that Mcm7 also binds promoters of early cell cycle genes not only in vitro but also in vivo, DNA cross-linked to Mcm1 and Mcm7 by formaldehyde was sheared and then immunoprecipitated (chIP) using antibodies specific to Mcm1-Myc (Fig. 4A, lane 2) or to Mcm7 (lane 3). HA antibodies were used as a negative control for mock chIP (lane 1). The coding region of ATP11 was used as a negative control for crosslinked DNA. Both Mcm1 and Mcm7 were cross-linked to the promoter sequences of MCM7, MCM5, and CDC6 but not ATP11.
To show a direct correlation between the presence of Mcm1 and Mcm7 at the promoters and their effect on the expression of the early cell cycle genes, we examined the transcript levels of MCM7, MCM5, and CDC6 in the mcm7-1 (Fig. 4B, a) and mcm1-1 strains (Fig. 4B, b). The transcript levels of these genes are increased by 24-fold in the mcm7-1 strain (Fig. 4C, a). In contrast, transcript levels of these genes are reduced by about half in the mcm1-1 strain (Fig. 4C, b). These results suggest that the mutant forms of these proteins exert opposing effects; mcm1-1 depresses and mcm7-1 stimulates ECB gene expression. This interpretation is consistent with the partial suppression of mcm7-1 by mcm1-1 in the mcm1-1 mcm7-1 double mutant (Fig. 1D).
Recruitment of Mcm7 to the ECB Promoters Occurs after the Onset of AnaphaseThe early cell cycle genes MCM7, MCM5, and CDC6 are expressed periodically during the M/G1 phase or early G1 phase. To correlate recruitment of Mcm7 to the MCM7 promoter with MCM7 expression, we followed these activities in a time course traversing the M/G1 phase. In addition, recruitment of Mcm7 to the promoters of MCM5 and CDC6 was also analyzed. Because Mcm7 is a subunit of the Mcm2-Mcm7 complex that is recruited to replication origins from late M to early S phase (25, 26), the association of Mcm7 with ARS305 DNA was used as a control.
An effective method for synchronizing yeast cells during the
M/G1 phase is to deplete cells of Cdc20, an activator of the
anaphase-promoting complex using the cdc20
(GAL-CDC20) strain
(46). Fluorescence-activated
cell sorting analysis shows that cells that have been arrested and then
released from the cdc20 block resume traversing the M and
G1 phase in synchrony up to about 105 min after release
(Fig. 5A). Based on
DNA content, >90% of the cells are in M phase 60 min after release from
block. At 75 min,
50% of the cells contain one genome-equivalent of DNA,
indicating the beginning of the G1 phase. At 90 and 105 min,
>90% cells are in G1 phase. Cell morphology indicates that
synchronous growth breaks down at about 105 min after release (data not
shown).
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The transcript level of MCM7 was quantified in a Northern blot
(Fig. 5B, panel
1) and expressed as a ratio of MCM7 RNA: ACT1 RNA
(Fig. 5C, panel
1). The transcript level of MCM7 peaks at two different points,
M phase (Fig. 5B,
panel 1, 0 min) and late G1 phase (105 min). The
MCM7 transcript level oscillates as much as 810-fold at its
peak and trough during the course of the cell cycle. The association of Mcm7
with the ECB promoters and ARS305 was measured as well by chromatin IP
(Fig. 5B, panels
25). Cross-linked DNA was PCR-amplified with
[
-32P]dATP and analyzed by PAGE. The amount of Mcm7
associated with the different promoter DNAs is quantified using a
PhosphorImager and plotted as the ratio of the PCR-amplified signal of each of
the promoter DNA to that of the ATP11 coding region
(Fig. 5C, panels
25). The strongest association of Mcm7 with the ECB promoters
occurs at the trough of the bimodal expression of MCM7 after the
initiation of anaphase. This pattern is reproducibly observed for Mcm7 at the
MCM7 (panel 2), MCM5 (panel 3), and
CDC6 (panel 4) promoters in independent experiments. In
contrast, Mcm7 is recruited to ARS305 in anaphase or telophase
(Fig. 5, B and
C, panels 5), slightly after its recruitment to
the ECB promoters, and remains associated with ARS305 throughout the
G1 phase.
Mcm3 Is Also Recruited to ECB PromotersTo investigate
whether other subunits of the MCM27 complex are also recruited to the
ECB promoters, we carried out chIP using antibodies specific to Mcm7 and Mcm3
(Fig. 6A). Extracts
were prepared from cells arrested in nocodazole, factor, and
hydroxyurea, respectively, to produce cell populations that are arrested in
different stages of M, G1, or S phase. Our results indicate that
Mcm3 and Mcm7 have very similar cell cycle localization patterns at respective
ECB promoters. Like Mcm7 (Fig.
6B), Mcm3 (Fig.
6C) is localized at the promoters of MCM7 and
MCM5 during M and G1 phase and slightly delayed at the
CDC6 promoter. The temporal localization patterns of Mcm7 in cells
synchronized by chemicals (Fig.
6) or by Cdc20 depletion (Fig.
5) are consistent within the resolutions of these approaches.
These results suggest that Mcm7 may bind ECB promoters in concert with other
subunits of the MCM27 complex in vivo.
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DISCUSSION |
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Mcm1 and Mcm7 Exert Opposing EffectsThe regulation of MCM7 expression by Mcm1 and Mcm7 and the interaction of these two proteins at the MCM7 promoter may explain the partial dosage suppression of mcm1-1 by MCM7 and mcm7-1 by MCM1. Indeed, similar relationships may also apply to Mcm1 and other subunits of the MCM27 complex. Mcm1 and Mcm7 appear to have opposite effects on the expression of early cell cycle genes: The mcm1-1 mutation reduces whereas the mcm7-1 mutation increases the expression of these genes. These phenotypes are consistent with Mcm1 acting as a positive regulator and Mcm7 a negative regulator in the expression of early cell cycle genes. The partial suppression of the growth defect of mcm7-1 by mcm1-1 in a double mutant (Fig. 1D) is consistent with this interpretation. The double mutant phenotype cannot be easily explained by the reduced expression of a functionally compromised Mcm7-1 protein.
The effect of Mcm7 as a repressor is apparent during the M/G1 phase when expression of MCM7 is induced and then quickly shut off (Fig. 5, B and C, panels 1). The time-course experiment shows that during M phase, when Mcm1 is bound to the promoter, MCM7 expression is induced (0 min). After the initiation of anaphase, a dampening of this expression coincides with the recruitment of Mcm7 to the promoter (panel 2, 15 and 30 min). A second burst of expression of MCM7 is observed in late G1 phase (panel 1, 90 and 105 min). A bimodal expression of early cell cycle genes involving Mcm1 and its coregulators has been reported for other early cell cycle genes including CDC6 (50) and CLN3 (29). Transcript levels of these genes reach a peak at M phase and again at late G1 phase. The bimodal transcription is believed to result from the coordinated action of multiple regulatory factors to fine tune expression of early cell cycle genes in response to multiple transducing signals (50). An examination of the MCM7 promoter sequence indicates that in addition to the tandem ECBs where Mcm1, Yox1, Yhp1 (19), and Mcm7 bind, a conserved early G1 element known as the SCB (Swi4/6 cell cycle box) is located between the ECBs and the translational start codon. SCBs are regulatory elements for early G1 gene expression (29, 51, 52). The bimodal expression of MCM7 could be the combined effects of ECBs and SCBs in addition to other factors.
Dual Roles of Mcm7 at Promoters and at Replication
OriginsParallels can be drawn between Mcm7, the transcription
factor, and Mcm7, a subunit of the replicative MCM helicase. Both are
recruited to their designated sites in a cell cycle-regulated manner at
approximately the same time frame. Mcm7 is recruited to the ECB promoters
after the initiation of anaphase upon release from the cdc20 block
(Fig. 7A). Recruitment
of Mcm7 to replication origins occurs 30 min later at anaphase or
telophase (Fig. 7B).
Neither Mcm7 alone nor the hexameric complex
(53,
54) exhibits DNA binding
activity, yet both are recruited by pre-assembled factors: Mcm1 at promoters
and other components of the pre-RC at replication origins
(55). Both Mcm7 and the MCM
complex are released from their respective binding sites after execution of
their respective functions
(25,
26). In both cases,
re-assembly at promoters or replication origins is prevented until the next
cell cycle.
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A number of reports support the view that Mcm7 as well as other subunits of
the MCM complex may participate in transcription regulation in mammals. Mcm7
directly interacts with the retinoblastoma tumor suppressor protein, for
example (56). Mcm7 also
interacts with MAT1, the targeting factor for the CAK kinase, substrates of
which include the cyclin-dependent kinases as well as components of the
transcription machinery such as the C-terminal domain of RNA polymerase II
(57). Stat1, an
interferon-
-responsive transcription factor, recruits a Mcm3/Mcm5
subcomplex through direct interaction with Mcm5 in interferon-
-induced
gene expression (58). In yet
another study, subunits of the MCM complex have been shown to associate with
the RNA polymerase II holoenzyme
(59). Our results suggest that
both Mcm7 and Mcm3 are recruited to the promoters of some ECB genes in a cell
cycle-specific manner in the budding yeast. It is possible that members of the
Mcm2Mcm7 family other than Mcm7 may also be involved in the regulation
of ECB gene expression.
Subtle Effects in Transcription May Have Significant Effects on
Replication InitiationA 2-fold reduction in the transcript levels
of MCM7 has a dramatic effect on minichromosome maintenance. This
observation is incongruent with the abundance of Mcm7 (30,000/cell)
(34), which far exceeds the
number of replication origins (
400/cell) in a yeast cell
(38,
60). Furthermore, this extreme
abundance of Mcm7 protein level appears not to be influenced by the
fluctuation of the MCM7 transcript levels
(31). However, dosage effects
are common among some of the MCM27 proteins
(32). That a small change in
the total pool of Mcm7 may produce a large effect on DNA replication is best
explained if a distinct pool of Mcm7 is targeted for DNA replication. A recent
study showed that a small subset of the Mcm3 population is polyubiquitinated
during M phase before the establishment of the pre-replication complex
(36). In that scenario, a
small nascent pool of the Mcm3 protein that is transcribed and translated in a
cell cycle-dependent manner may be targeted for replication initiation.
Autoregulation of replication initiation factors effectively limits DNA
replication to a narrow window by producing a burst of the initiation factors
just prior to replication initiation. This strategy is used in the regulation
of the E. coli DnaA protein
(61) and the SV40 large T
antigen (62,
63).
Coordination of Gene Expression and Cell Proliferation by Common FactorsThere are two issues that a eukaryote cell must contend with in cell growth and cell proliferation: the execution of DNA replication and gene expression, and the coordination of these two processes. Here, we show that Mcm7, a subunit of the putative replicative helicase that is involved in the execution of DNA replication, may have a role in the expression of DNA replication genes. Similarly, Mcm1, which regulates expression of a number of replication initiation factors, also binds replication origins (21). By involving the same protein factors in the execution of both of these processes, cell growth and cell proliferation may be coordinated. A paradigm of this complex scheme can be found in the large T antigen of the SV40 virus, a successful parasite that has evolved to infiltrate the infrastructure of its host (64). Of particular relevance, as a smaller unit, the large T antigen is a transcription factor (65); as a hexamer, it is a replicative helicase (66). Understanding the dynamics between the hexameric MCM helicase and its subunits in the regulation of DNA replication and gene expression will provide insights into the coordination of cell growth and cell proliferation in eukaryotes.
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FOOTNOTES |
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Current address: Scripps Research Inst., La Jolla, CA 92037.
Supported by National Institutes of Health Predoctoral Training Grant
GM07273.
¶ Graduate Assistance in Areas of National Need fellow.
|| To whom correspondence should be addressed. E-mail address: bt16{at}cornell.edu.
1 The abbreviations used are: ORF, open reading frame; GST, glutathione
S-transferase; BSA, bovine serum albumin; SCB, Swi4/6 cell cycle box;
chIP, Chromatin immunoprecipitation; ECB, early cell cycle box; HA,
hemagglutinin; MCE, Mcm1 consensus element.
2 S. DiTusa, M. Osman, and B. Tye, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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