Isolation of a stable Cdc45-MCM complex containing hyper-phosphorylated Mcm4 exclusively on the S-phase chromatin
To understand how Cdc45 is recruited to chromatin during S phase, we examined components associated with Cdc45 on the S-phase chromatin. Log-phase yeast cells were arrested in G1 phase using α-factor and in S phase using hydroxyurea (HU). A large-scale chromatin fractionation procedure (), modified from a previous protocol was used (Liang and Stillman, 1997
). Crude yeast extract was separated into a soluble protein and another fraction containing chromatin-bound proteins released after exhaustive DNase I digestion of the chromatin pellet (SN2). Cdc45 was immunoprecipitated from SN2 using polyclonal antibody against Cdc45. Analysis of the Cdc45 immunoprecipitate (IP) from the S-phase SN2 revealed several specific protein bands that were absent in the Cdc45 IP from the G1 SN2, in which Cdc45 was not present, or in the control IP using 12CA5 antibody (. lanes 2 & 3). Mass spectrometry analysis demonstrated that these bands correspond to Cdc45 and all six MCM subunits. This result was confirmed by immuno-blot analyses (, middle panel and data not shown). The stoichiometry of Cdc45 and each MCM subunits is not known, however, the intensity of each band in the silver-stained gel was comparable. In addition, the yeast Mcm3, Mcm5 and Mcm7 proteins individually co-purified with the yeast Cdc45 when co-expressed in the insect cells (unpublished results), suggesting direct interaction of Cdc45 with multiple MCM subunits.
MCM proteins were not detectable in the Cdc45 IP of SN1 fraction prepared from G1 and S-phase cells even though all the components are abundant (, left and middle panels). Moreover, the complex was not present on G1-phase chromatin since Cdc45 was not there. Thus the stable Cdc45-MCM interaction occurs only on chromatin and only during S phase. These observations suggest that assembly of the Cdc45-MCM complex is a regulated process. Interestingly, a portion of total Mcm4 on the S-phase chromatin was modified and exhibited a slower mobility in SDS polyacrylamide gel electrophoresis (SDS-PAGE) (, left panel). The slower form of Mcm4 was greatly enriched after Cdc45 IP (, right panel). Lamda phosphatase treatment of the Cdc45 IP demonstrates that this modification was phosphorylation (). The mobility shift was prominent considering the size of the protein (~110 kDa) and the phosphatase treatment sometimes resulted in multiple intermediate bands, suggesting that the Mcm4 subunit in the Cdc45-MCM complex isolated from S-phase arrested cells is hyper-phosphorylated on multiple sites.
The Cdc45-MCM complex containing hyper-phosphorylated Mcm4 in an unperturbed S phase
Because the HU-induced arrest in S phase also triggers a DNA damage checkpoint response, it raises the concern that hyper-phosphorylation of Mcm4 is a response to HU treatment rather than a normal S-phase event. To address this issue, yeast cells were arrested in G1 using α-factor and then released into fresh YPD medium. Cells were collected at different times after release and fractionated for chromatin-bound proteins (). Immuno-blot analysis showed that a small portion of Mcm4 was hyper-phosphorylated as cells entered S phase (about 40–60 min after release), as measured by budding index and CDK-dependent phosphorylation of Orc6 (Liang and Stillman, 1997
; Nguyen et al., 2001
). Hyper-phosphorylation of Mcm4 occured concomitant with Cdc45 binding to chromatin. Cdc45 IP of SN2 prepared from the time-course samples co-precipitated the MCM complex containing mainly the hyper-phosphorylated Mcm4 in S phase (). Thus, hyper-phosphorylated Mcm4 was specifically present in the Cdc45-MCM complex on chromatin during normal cell cycle progression in S-phase.
The role of DDK in hyper-phosphorylation of Mcm4 and Cdc45-MCM complex formation during S phase
Because DDK is required for Cdc45 loading and origin activation and purified DDK can phosphorylate Mcm4 in vitro
(Lei et al., 1997
; Weinreich and Stillman, 1999
; Zou and Stillman, 2000
), we asked whether DDK was required for hyper-phosphorylation of Mcm4 on chromatin and Cdc45-MCM complex formation during S phase. Although the essential role of DDK can be bypassed in the mcm5-bob1
mutant (Hardy et al., 1997
), mcm5-bob1 cdc7Δ
cells proliferate much slower than mcm5-bob1
cells (Weinreich and Stillman, 1999
); ), suggesting a partial bypass. Flow cytometry analysis of DNA content in cells released from G1 arrest showed that, compared with congenic mcm5-bob1
cells, mcm5-bob1 cdc7Δ
cells were defective in S-phase entry and progression (Weinreich and Stillman, 1999
); ). We isolated SN2 from mcm5-bob1
and mcm5-bob1 cdc7Δ
cells arrested in HU. Because mcm5-bob1 cdc7Δ
cells are larger than mcm5-bob1
cells, same volume of mcm5-bob1 cdc7Δ
cell pellet contained roughly half the number of mcm5-bob1
cells. When same amount of total SN2 protein from each of mcm5-bob1
and mcm5-bob1 cdc7Δ
cells was compared by immunoblot analysis, similar amounts of Mcm4, Mcm2, Mcm3 and Cdc45 were detected (). While a small portion of Mcm4 was hyper-phosphorylated in SN2 from mcm5-bob1
cells, hyper- phosphorylation of Mcm4 was not detectable in SN2 from mcm5-bob1 cdc7Δ
cells (). The presence of Cdc45 in the mcm5-bob1 cdc7Δ
chromatin is consistent with the idea that mcm5-bob1
mutation allows Cdc45 loading independent of DDK (Sclafani et al., 2002
). However, when Cdc45 from each of these SN2 fractions was immunoprecipitated, much less MCM subunits were present in the purification from mcm5-bob1 cdc7Δ
chromatin. This result suggests that, in the absence of DDK activity, the Cdc45-MCM complex is not formed efficiently or is less stable. Thus, DDK plays an important role in regulating Mcm4 phosphorylation and proper Cdc45-MCM interaction in vivo
, in addition to promoting S-phase progression.
CDC7 is required for normal S-phase progression, hyper-phosphorylation of Mcm4 in vivo and stable Cdc45-MCM complex formation. Strains YS814, YS819, YS824 and YS828 are used here
Identification of regions in Mcm4 important for phosphorylation by DDK
As illustrated in , a major part of Mcm4 is very conserved among orthologs and contains distinct motifs of the AAA+ ATPase family and zinc finger motifs that are likely involved in double-hexamer formation (reviewed in Forsburg, 2004
; Labib and Diffley, 2001
; Tye and Sawyer, 2000
). In contrast, the N-terminus of Mcm4 is not conserved in primary sequence, but is extremely S/T rich in all eukaryotic Mcm4 orthologs (29% in S. cerevisiae
Mcm4). We thus term this region NSD (for N
In vitro phosphorylation of Mcm4 fragments using purified DDK
To determine which region of Mcm4 is important for phosphorylation, various Mcm4 fragments were expressed and purified from bacteria and used as substrates for the yeast DDK expressed in and purified from insect cells (; in this article, substrates refer to protein substrates for the kinase; substrate ATP in this study is constant at 100 μM). Mcm4 fragments without NSD (mcm4175–933 and mcm4175–333) were not phosphorylated to a detectable level, indicating that NSD was essential for phosphorylation. NSD (mcm41–175) alone was phosphorylated by DDK albeit much less efficiently than the full-length protein (, compare substrates 1 and 2). Nevertheless, this result indicateed that NSD contained DDK phosphorylation sites. Interestingly, mcm41–333 was phosphorylated as efficiently as the full-length protein (compare substrates 1 and 4), suggesting that, in addition to NSD, an additional element within amino acid residues 175–333 was important for efficient phosphorylation.
Notably, most of the 32P incorporation in mcm41–333 migrated slower than the bulk of the mcm41–333 protein in SDS-PAGE. The mobility shift was substantial and reminiscent of the Mcm4 protein shift in the Cdc45-MCM complex isolated from yeast chromatin. The shift is most likely due to high stoichiometric phosphorylation (i.e. multiple phosphorylation on a single molecule). Hence, we refer this shifted band as the hyper-phosphorylated form and the band co-migrating with the input protein as hypo-phosphorylated.
mcm4146–333, which contains a small portion of NSD, was phosphorylated efficiently. Mutation of multiple S/T to A in this portion of NSD (mcm4146–333, 5A+2A) abolisheed the phosphorylation, consistent with the presence of phospho-acceptor sites at the NSD. In contrast to mcm41–333, phosphorylation of mcm4146–333 did not result in a mobility shift. Thus, hyper-phosphorylation required an extended NSD.
Taken together, we identified two regions important for defining Mcm4 as the substrate of DDK. The NSD contains phospho-acceptor sites for DDK and an element in the conserved region next to NSD facilitates efficient phosphorylation, but is not itself a phosphorylation target.
Processive phosphorylation of Mcm4 by DDK
Phosphorylation of mcm41–333
produced two major bands of distinctive gel mobility and few intermediate bands, suggesting that the kinase and the substrate did not dissociate after each phosphorylation event before the completion of stoichiometric phosphorylation (Duyster et al., 1995
). Thus, hyper-phosphorylation of mcm41–333
was likely processive.
To further address the mechanism of hyper-phosphorylation, kinase assays were performed using various substrate concentrations (). If phosphorylation was distributive, rather than processive, increasing the concentration of the substrate should result in a decrease in hyper-phosphorylation because the high concentration of unphosphorylated substrate would compete with the partially phosphorylated substrates each time substrate and enzyme dissociate (Burack and Sturgill, 1997
). In contrast, increasing the concentration of mcm41–333
in the kinase assay did not cause a decrease in the amount of the hyper-phosphorylated product (), consistent with non-distributive phosphorylation. Only when substrate concentration was near or lower than the enzyme concentration did the ratio of the two phosphorylated products change (), suggesting that the hyper-phosphorylated form was produced from processive phosphorylation as a result of high affinity interaction between the substrate and the kinase through an additional interaction distinct from that of the catalytic site and the phospho-acceptor.
Processive phosphorylation of mcm41-333 through a kinase docking mechanism
mcm4175-333 binds to DDK
To determine whether Mcm4 binds to DDK, as is implied by the processive nature of phosphorylation, we expressed and purified various GST fusions of mcm4 fragments to test binding to purified DDK in vitro. Using a GST-pull down assay, GST-mcm41-333, GST-mcm4175–333, GST-mcm4146–333, and GST-mcm4146–333, 5A+2A, but not GST or GST-mcm41–175 bound to purified DDK (). These interactions occurred both in the presence or absence of ATP (data not shown). In addition, GST-mcm4175–333, GST-mcm4146–333 and GST-mcm4146–333, 5A+2A interacted with DDK, suggesting that DDK phosphorylation sites were not required for the interaction. Therefore, Mcm4 bound to DDK through an element within amino acid residues 175–333.
A conserved region adjacent to NSD of Mcm4 involved in DDK recruitment
The fact that mcm4175–333 bound DDK () and functions in cis, but not in trans, for efficient phosphorylation of NSD ( and data not shown) strongly suggested that this element promoted phosphorylation by binding to the kinase. This idea predicted that, if added in trans, mcm4175–333 could compete with mcm41–333 for binding to DDK and thereby interfere with its phosphorylation.
To test the prediction, the phosphorylation of mcm41–333 in the presence of various amounts of mcm4175–333 was analyzed. In the kinase assay using 30 nM kinase and 0.25 μM mcm41–333, mcm4175–333 inhibited phosphorylation of mcm41–333 in a concentration-dependent manner (, lanes 7–11). The inhibition was evident even at 0.25 μM of mcm4175–333 fragment (compare lanes 6 and 7). In contrast, auto-phosphorylation on the Dbf4 subunit was not inhibited, but rather, increased in the presence of mcm4175–333, suggesting that the inhibitory effect of mcm4175–333 was specific to mcm41–333. The functional significance of auto-phosphorylation and its stimulation require further studies. Unlike mcm4175–333, NSD fragment (mcm41–175) only caused minor inhibition on the phosphorylation of mcm41–333 and only at high concentrations (, lanes 1–5), probably due to competition between phospho-acceptors for the catalytic site on DDK.
These results suggest that mcm4175–333 contains a DDK-docking domain (DDD) and, together with phospho-acceptor sites within NSD, define Mcm4 as a specific substrate for DDK. As an added proof, we are able to create an artificial DDK substrate by attaching a heterologous phospho-acceptor sequence to the N-terminus of DDD (, substrate 9).
DDK recruitment by DDD accounts for processive phosphorylation at NSD
The kinase docking model also provided mechanistic explanation for the processive, high stoichiometric phosphorylation of mcm41–333 by DDK. This model predicted that mcm4175–333 should have little effect on the high stoichiometric phosphorylation of mcm41–333 if the DDK-mcm41–333 complex was allowed to form first. The inhibitory effect was eliminated when mcm41–333 was pre-incubated with DDK before mcm4175–333 and ATP were added (, compare lane 7 to lanes 8–12). For unknown reasons, pre-incubation reduced the overall phosphorylation. Nevertheless, addition of excess amounts of mcm4175–333 had little effect on hyper-phosphorylation of mcm41–333. This result argued that DDD-kinase interaction was the underlying mechanism for processive phosphorylation of NSD by DDK.
To further attribute elements in DDD that promote NSD phosphorylation, deletions of DDD from the C-terminus of mcm41–333 were prepared for in vitro kinase assay. Serial deletions down to mcm41–200 did not result in a significant decrease in phosphorylation levels (). Rather, as the DDD region shortened, the relative levels of high stoichiometric phosphorylation decreased, along with the accumulation of bands corresponding to phosphorylation of intermediate stoichiometry, suggesting decreased processivity. Thus, shortening the DDD resulted in weaker binding of enzyme to substrates. Even though mcm41–200 has only 25 more additional residues than the NSD fragment (mcm41–175), it was phosphorylated much more efficiently than NSD alone. Therefore, residues 176–200 might constitute part of the DDK docking sites but additional sequences are needed for more efficient recruitment of DDK.
NSD is important for the function of Mcm4 in vivo
To determine whether the NSD is important for the biological function of Mcm4, we constructed various alleles of MCM4 with truncations of the NSD region and tested whether these mutants could rescue the lethality of mcm4Δ using a plasmid shuffle assay (). A mutant, mcm4Δ2-174, which lacked the NSD, rescued mcm4Δ poorly. In contrast, mutant alleles with partial NSD (mcm4Δ2-73, mcm4Δ74-174, mcm4Δ2-112 and mcm4Δ2-145) rescued mcm4Δ as well as the full-length MCM4, suggesting that NSD, although dispensable, is important for the fitness of cells. The fact that either mcm4Δ74-174 or mcm4Δ2-73 can function normally in vivo suggests that residues 2-73 (20 S/T residues) and residues 74-174 (31 S/T residues) are redundant. Moreover, one of the alleles, mcm4Δ2-145, which supports normal cell growth, has only 29 residues (146–174) of NSD, with 10 S/T residues. The functional redundancy can explain why the primary sequence of NSD, apart from being S/T rich, is not conserved among Mcm4 orthologs.
NSD is important for normal cell growth and S-phase progression
The N-terminus of Mcm2 can functionally replace the NSD of Mcm4
The divergent nature of NSD prompted us to investigate whether phosphorylation sites from another DDK substrate can functionally replace NSD. Like Mcm4, Mcm2 has been shown to be a DDK substrate in vitro
(Lei et al., 1997
; Weinreich and Stillman, 1999
). Genetic interaction between MCM2
has suggested Mcm2 as a likely physiological target for DDK (Lei et al., 1997
). However, the functional significance of Mcm2 phosphorylation was not demonstrated. To test whether potential DDK target sites from Mcm2 could function in the context of Mcm4, we replaced the Mcm4 NSD with the N-terminal 200 residues of Mcm2 to generate the allele mcm21-200-mcm4Δ2-174
. This fusion protein functionally replaced Mcm4 in vivo
(). In addition, the fusion protein mcm21-200
was a good substrate for DDK in vitro
(data no shown). Further phenotypic analysis showed that the fusion protein functioned as well as the wild-type Mcm4 in supporting normal cell growth and timely S-phase progression (see below; ). These results further strengthen the idea that phosphorylation of multiple MCM components by DDK is of physiological importance.
The NSD of Mcm4 is important for normal cell growth and S-phase progression
The phenotypes of cells containing different alleles of MCM4 were further investigated. Two independent isolates of mcm4Δ2-174 cells grew very slowly in liquid YPD media at 30oC, compared with cells harboring the wild-type MCM4 (). In contrast, the growth curves of mcm4Δ2-145 and mcm21-200-mcm4Δ2-175 cells were very similar to that of the wild type. On YPD plates, the two mcm4Δ2-174 isolates grew slowly at 25oC, 30oC and 37oC and did not grow at 15oC and 16oC (, and data not shown) while mcm4Δ2-73, mcm4Δ74-174, mcm4Δ2-112, mcm4Δ2-145 and mcm21-200-mcm4Δ2-175 cells grew just like wild-type cells at those temperatures. Thus, mcm4Δ2-174 cells were cold-sensitive.
DDK phosphorylation sites at the N-terminus of Mcm4 are important in vivo
Flow cytometry analysis of asynchronous cell populations revealed that mcm4Δ2-174 cells accumulated more S-phase cells compared to MCM4 and mcm21-200-mcm4Δ2-175 cells (). After treatment with HU, MCM4 and mcm21-200-mcm4Δ2-175 cells arrested mostly in the early S phase, while mcm4Δ2-174 cells arrested at all stages across the S phase, displaying a broader peak between 1C and 2C DNA content. This result suggested that mcm4Δ2-174 cells take more time going through S phase than MCM4 and mcm21-200-mcm4Δ2-175 cells, explaining why HU induced a broad S phase pattern of DNA content.
To further assess the S-phase progression of these cells, we arrested cells in G1 using α-factor, released them into fresh medium and collected cells for flow cytometry analysis at indicated time points (). While MCM4
cells were well into S phase 30 min after release and entered the next G1 by 105 min, mcm4Δ2-174
cells just entered S phase at 45 min and progressed slowly across S phase and most cells still did not reach the next G1 by 150 min. The delayed entry and progression of S phase is reminiscent of what was observed for mcm5-bob1 cdc7Δ
cells (Weinreich and Stillman, 1999
); ). Taken together, NSD of Mcm4 is essential for timely entry and progression through S phase.
DDK phosphorylation sites at the N-terminus of Mcm4 are important in vivo
Since mcm4Δ2-145 was able to rescue mcm4Δ as efficiently as the full-length Mcm4, we asked whether the remaining 10 S/T residues in this region were important for the function of the protein. Different combinations of S/T to A mutations were made in this short NSD () and their ability to rescue mcm4Δ determined (). Mutation alleles mcm4Δ2-145, 2A and mcm4Δ2-145, 3A+2A rescued mcm4Δ as efficiently as mcm4Δ2-145 while mcm4Δ2-145, 5A functioned less than mcm4Δ2-145 but better than mcm4Δ2-174. Mostly noticeably, mcm4Δ2-145, 5A+2A, which has 7 S/T to A mutations, rescued mcm4Δ as poorly as mcm4Δ2-174. Like mcm4Δ2-174, mcm4Δ2-145, 5A and mcm4Δ2-145, 3A+2A exhibited a cold-sensitive phenotype and did not grow at 15oC (, sectors 5 and 6). Moreover, as shown earlier in the in vitro kinase assay (), an mcm4 fragment containing the same short NSD as mcm4Δ2-145 (mcm4146-333) was phosphorylated efficiently by DDK while the substrate with the same 7 S/T to A mutations (mcm4146-333, 5A+2A) was not. Thus, the in vivo function of the Mcm4 N-terminus correlates with its capacity as a DDK substrate.
Unexpectedly, addition of the 3xHA sequence, which contains no S/T residues, to the N-terminus of mcm4Δ2-174 abolished the function of the protein (). We asked whether re-introducing serine residues into this heterologous sequence might allow phosphorylation of the resulting fusion protein SS/HA-mcm4Δ2–174 () and thereby render it functional. Indeed, SS/HA-mcm4Δ2-174 not only functionally complemented mcm4Δ, but it grews better than mcm4Δ2-174 and was rescued from the cold-sensitive phenotype (, sectors 7 and 8). In addition, in vitro kinase assay demonstrated that SS/HA-mcm4175-333 was phosphorylated by DDK while 3xHA-mcm4175–333 was not (, substrates 8 and 9). Taken together, these data demonstrate that phosphorylation of Mcm4 at its N-terminus by DDK is required for cell growth.
Deregulation of Cdc45 is detrimental in NSD mutants of MCM4
Since phosphorylation of Mcm4 by DDK is important for cell growth and DDK is required for formation of the stable Cdc45-MCM complex on the S phase chromatin, it is likely that these processes are functionally linked. Such link may be revealed in by synthetic genetic interactions.
We examined the effect of Cdc45 overproduction in MCM4, mcm4Δ2-174, mcm4Δ2-145 and mcm4Δ2-145, 3A+2A cells. Control vector derived from a CEN-based plasmid, pRS416, carrying only GAL1,10 promoter sequence or the vector containing GAL1,10::CDC45 were introduced into these cells. On the SC-URA plate, where the GAL1,10 promoter was repressed, MCM4, mcm4Δ2-174, mcm4Δ2-145 and mcm4Δ2-145, 3A+2A cells carrying either vectors grew similarly to each other (, left panel). On the SC-URA-Gal plate, where the GAL1,10 promoter is induced, MCM4 and mcm4Δ2-145 cells carrying either vectors also grow similar to each other (, right panel, compare sectors 1 and 2 and sectors 5 and 6), indicating that overproduction of Cdc45 did not affect the growth of these cells. In contrast, on the same plate, mcm4Δ2-174 and mcm4Δ2-145, 3A+2A cells carrying GAL1,10::CDC45 grew poorly and formed micro-colonies that eventually stopped growing, while cells harboring control vectors grew just fine (, right panel, compare sectors 3 and 4 and sectors 7 and 8). Thus, overproduction of Cdc45 impairs the growth of NSD mutants in MCM4. This genetic interaction, is consistent with the hypothesis that phosphorylation of the MCM complex by DDK is important for the regulated assembly or activity of the Cdc45-MCM complex.
Phosphorylation of MCM by DDK may be important for proper engagement of Cdc45 to the MCM complex