Download 1 MCM10 MEDIATES THE INTERACTION BETWEEN DNA

Genetics: Published Articles Ahead of Print, published on December 8, 2008 as 10.1534/genetics.108.099101
MCM10 MEDIATES THE INTERACTION BETWEEN DNA REPLICATION AND
SILENCING MACHINERIES
Ivan Liachko and Bik K. Tye*
Department of Molecular Biology and Genetics
Cornell University
327 Biotechnology Building
Ithaca, NY 14853
Running Title: MCMs in Heterochromatic Silencing
Abbreviations: MCM – Minichromosome Maintenance; ORC – Origin Recognition
Complex; pre-RC – Pre-Replication Complex; SIR – Silent Information Regulator
* corresponding author: Bik K. Tye; 325 Biotechnology Building, Ithaca, NY 14853;
[email protected]; 607-255-2445
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ABSTRACT
The connection between DNA replication and heterochromatic silencing in yeast
has been a topic of investigation for over twenty years. While early studies showed that
silencing requires passage through S-phase and implicated several DNA replication
factors in silencing, later works showed that silent chromatin could form without DNA
replication. In this study we show that members of the replicative helicase (Mcm3 and
Mcm7) play a role in silencing and physically interact with the essential silencing factor,
Sir2, even in the absence of DNA replication. Another replication factor, Mcm10,
mediates the interaction between these replication and silencing proteins via a short Cterminal domain. Mutations in this region of Mcm10 disrupt the interaction between Sir2
and several of the Mcm2-7 proteins. While such mutations caused silencing defects, they
did not cause DNA replication defects or affected the association of Sir2 with chromatin.
Our findings suggest that Mcm10 is required for the coupling of the replication and
silencing machineries to silence chromatin in a context outside of DNA replication
beyond the recruitment and spreading of Sir2 on chromatin.
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INTRODUCTION
Large regions of eukaryotic genomes are packaged into transcriptionally silent
heterochromatin. Yeast heterochromatic silencing is established and maintained by the
action of a group of factors called silent information regulators (SIRs) (RUSCHE et al.
2003). Sir2, Sir3 and Sir4 are recruited to chromatin and spread bi-directionally in a
stepwise fashion until encountering a boundary element (HOPPE et al. 2002; RUSCHE et
al. 2002; THON et al. 2002). The silencing activity of these proteins is attributed to the
histone deacetylase function of Sir2, although Sir3 and Sir4 are also required for
silencing (IMAI et al. 2000). Silencing in the budding yeast Saccharomyces cerevisiae is
largely limited to telomeres, the silent mating type loci, and rDNA. In telomeres the SIRs
are recruited to chromatin by Rap1 (KYRION et al. 1993; MORETTI et al. 1994). In the
silent mating type loci (HML and HMR) the binding and spreading of SIRs is initiated by
the combined action of the Origin Recognition Complex (ORC), Rap1, and Abf1 binding
to DNA elements termed silencers (RUSCHE et al. 2003). Once formed, this
transcriptionally silent epigenetic structure can be stably inherited for up to 40
generations (PILLUS and RINE 1989).
An early study in the cell-cycle regulation of silent chromatin showed that
passage through S-phase was required for the establishment of silencing (MILLER and
NASMYTH 1984), suggesting that DNA replication is involved in silencing. Indeed,
several members of the replication machinery, such as ORC, Mcm10, Mcm5, Cdc7,
Abf1, and PCNA have been since implicated in silencing and chromatin structure
(AXELROD and RINE 1991; BELL et al. 1993; BURKE et al. 2001; CHRISTENSEN and TYE
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2003; DZIAK et al. 2003; EHRENHOFER-MURRAY et al. 1999; LIACHKO and TYE 2005;
MCNALLY and RINE 1991; ZHANG et al. 2000). However, several studies have shown
that DNA replication is not required for the establishment of silencing (KIRCHMAIER and
RINE 2001; LAU et al. 2002; LI et al. 2001; MARTINS-TAYLOR et al. 2004). A more
recent study showed that recruitment of Sir proteins to chromatin is a necessary but is not
the final step for the establishment of silencing which may be completed as late as M
phase (KIRCHMAIER and RINE 2006).
One key component of DNA replication machinery is the pre-Replication
Complex (pre-RC) which assembles on replication origins in late-M/early-G1 phases of
the cell cycle prior to the initiation of DNA replication at the beginning of S phase. The
pre-RC consists of a large number of proteins such as Orc1-6, Cdc6, Cdt1, and the
replicative helicase Mcm2-7 complex (FORSBURG 2004). The Mcm2-7 complex consists
of six minichromosome maintenance (MCM) proteins (FORSBURG 2004; TYE 1999). One
distinct characteristic of the MCM2-7 family is a conserved domain known as the MCM
box, which spans about 200 residues near the center of the protein (TYE and SAWYER
2000). The MCM box includes two ATPase motifs, the Walker A motif and the Walker
B motif, as well as an arginine-finger motif. In addition to these features, all six of the
MCM2-7 proteins, with the exception of Mcm3, have zinc binding motifs near the N
terminal regions, and some have nuclear localization signal (NLS) sequences and sites of
phosphorylation by cyclin dependent kinases (CDKs) (ISHIMI 1997; LEE and HURWITZ
2000). The MCM2-7 proteins are highly abundant proteins (estimated at more than
30,000 copies per cell in S. cerevisiae) whose levels are stable during the cell cycle
(FORSBURG 2004; LEI et al. 1996). In S. cerevisiae, MCM complexes outnumber the
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approximately 400 DNA replication origins by a factor of 75. The reason for this vast
overabundance is unclear and only a small subset of the MCM2-7 proteins is associated
with chromatin even during the G1-S transition, when their chromatin association is at its
peak. Interestingly, reducing the levels of the MCM2-7 proteins causes defects in genetic
stability, suggesting that the extra protein molecules are necessary for a function that is
yet unknown (LEI et al. 1996; LIANG et al. 1999).
Mcm10 is an essential factor (MERCHANT et al. 1997) that is closely associated
with the Mcm2-7 complex although it is not part of the same protein family. Much like
the MCM2-7 proteins, it is highly abundant in the cell (KAWASAKI et al. 2000). Mcm10
stabilizes the Polα-primase complex (RICKE and BIELINSKY 2004; RICKE and BIELINSKY
2006; YANG et al. 2005) and is important for mediating interactions between other
replication proteins (DAS-BRADOO et al. 2006; LEE et al. 2003). Temperature sensitive
mutations in MCM10, mcm10-1 (P269L) and mcm10-43 (C320Y), cause multiple defects,
including loss of interactions with other proteins, defects in plasmid replication, and
pausing of replication forks at semi-permissive temperature (HOMESLEY et al. 2000;
MERCHANT et al. 1997). At restrictive temperature, mcm10 cells arrest at the end of S
phase with aberrant DNA structures (KAWASAKI et al. 2000; MERCHANT et al. 1997).
Recently, Mcm10 has been implicated to function in chromatin structure in yeast as well
as Drosophila melanogaster (CHRISTENSEN and TYE 2003; DOUGLAS et al. 2005;
LIACHKO and TYE 2005). In Drosophila, Mcm10 interacts with HP-1, an important
heterochromatin protein (CHRISTENSEN and TYE 2003), while in yeast Mcm10 interacts
with Sir2 (DOUGLAS et al. 2005; LIACHKO and TYE 2005). In addition, genetic
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experiments suggest that the silencing function of Mcm10 is separate from its replication
function (DOUGLAS et al. 2005; LIACHKO and TYE 2005).
In this study we show that several members of the MCM2-7 complex play a role
in heterochromatic silencing. In addition, they physically interact with Sir2, even in the
absence of DNA replication. Mcm10 is required for the interactions between Sir2 and
MCM2-7. We have localized the Mcm10 domain responsible for the interaction with
Sir2 to a 53-amino acid domain in the C-terminus of Mcm10. Mutations in this region
inhibit Mcm10-Sir2 interactions as well as the interaction of Sir2 with members of the
MCM2-7 family. These mutants also exhibit defects in silencing, but not in DNA
replication. Interestingly, mcm2-7 and mcm10 mutations that have a significant effect on
both DNA replication and silencing do not affect the association of Sir2 with chromatin.
Our findings show that MCM2-7 proteins have a silencing function which requires a
coupling of the replication and silencing machineries via Mcm10.
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MATERIALS AND METHODS
Strains and plasmids.
Strains used in this study are listed in Table 1. All strains are isogenic derivatives
of W303-1A, unless otherwise indicated. All procedures were performed according to
standard yeast methodology (SHERMAN 1991). Strains carrying silencing reporters were
made by crossing strain YB541 or YB697 to the appropriate mutant strain and selecting
desired segregants by their conditional phenotypes and/or auxotrophy. Genotypes were
confirmed by PCR or by plasmid complementation where applicable.
Plasmids used in this study are listed in Table 2. Plasmids used for the expression
of two-hybrid fusions were constructed by the Gateway system (Invitrogen). Gateway
cassettes were ligated into plasmids pBTM116 and pGAD2F, creating pBTMgw and
pGADgw respectively. pDONR201 entry clones containing MCM10 and SIRs ready for
N-terminal fusions were constructed according to Invitrogen instructions, and sequenced.
LR recombination reactions (Invitrogen) were set up between pGBT9gw, pGADgw,
pBTMgw, or pGBKgw and each of the aforementioned entry clones. These are
recombination reactions which replace the Gateway cassette in the relevant vector with
the gene from the entry clone. The full length pBTM-MCM10 was described in
(MERCHANT et al. 1997). All yeast transformations were carried out using standard
lithium acetate protocols (ORR-WEAVER et al. 1981; SHERMAN 1991).
Point mutations were introduced using a fusion-PCR (HORTON et al. 1989)
mutagenesis method. The DNA region to be mutagenized was PCR amplified in two
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separate fragments which overlap by 50 – 60 base pairs. The overlap primers contained
the desired mutation. After the initial PCR, the two fragments were purified separately
and used together in another PCR reaction without any template DNA or primers. The
overlapping regions in the two DNA fragments acted as primers for each other and PCR
produced a final molecule which contained the entire gene fragment including the
mutation of interest. This fragment was then cloned into the relevant vector.
To create the strains with tagged proteins, the SIR2-3HA and MCM10-13MYC
alleles were crossed out of strains WCY15, WCY39 (this lab) and ROY1515 (R.
Kamakaka). These were then crossed to make strains which have both alleles. Cterminal mutations were introduced into these strains by standard homologous gene
replacement methodology (ORR-WEAVER et al. 1981). The last 68 amino acids from the
C-terminus of MCM10 were deleted and replaced with HIS3 through one-step gene
replacement with a PCR product containing the HIS3 gene flanked by appropriate
MCM10 homology regions.
Silencing assays.
Yeast strains bearing the URA3 reporter were grown overnight in appropriate
dropout media. Tenfold serial dilutions were setup in sterile 96-well plates and a
constant volume of each dilution was spotted onto the appropriate plate using a multichannel pipettor. 5-FOA was used at a concentration of 1mg/ml. For experiments using
the hmr::ADE2 color reporter, strains were streaked out on rich media (YPD) plates,
grown at 30°C for 2-3 days, then placed at 4°C for 3 days for further color development.
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Yeast two-hybrid.
pGAD2F and pBTM116 constructs were transformed into the two-hybrid strain
EGY40 carrying the pSH18-34 reporter plasmid (FIELDS and SONG 1989). Interactions
were assessed by the appearance of blue colonies on plates containing Xgal (Sigma). 10
microliters of a relevant saturated culture were spotted onto X-gal plates and
photographed after 2-4 days of growth at 30°C.
Minichromosome maintenance (MCM) assays
MCM assays were performed exactly as described in (DONATO et al. 2006) using
the plasmid YCp1.
Chromatin immunoprecipitation (ChIP)
ChIP experiments were performed as previously described (GOLDFARB and ALANI
2004) using exponentially growing cultures at 30°C. Cultures of appropriate cells were
grown to log phase and then the cells were lysed in buffer containing 50mM HEPES,
1mM EDTA, 140mM NaCl, 1%Triton X-100, 0.1% NaDOC and a mix of protease
inhibitors as described. 5 micrograms of commercially available anti-HA antibody was
used for each immunoprecipitation (Roche 12CA5 cat#:11583816001). Protein G
Agarose beads (Roche cat#: 1719416) were used. The immunoprecipitated DNA was
analyzed with real-time PCR using SYBR Green PCR Master Mix from Applied
Biosciences (4309155) on the DNA Engine Cycler (PTC-200) with Opticon Detector
(CFD-3200) form MJ Research according to the manufacturer’s instructions.
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Co-immunoprecipitation (Co-IP)
Co-IP experiments were performed using the same protocol as ChIP with the
following differences. For experiments using DNAseI, the lysis buffer was changed to
contain no EDTA, which was replaced with 5mM MgCl2. Cells were arrested in G2/M
phase using 15μg/mL of nocodazole. The DNAseI used was from Invitrogen (cat#:
18068-015) and the samples were digested for 20 minutes at 37°C with 20U/mL of the
enzyme. After the lysates were incubated with the beads and the beads were washed
(same as ChIP protocol), the beads were boiled in SDS buffer containing DTT (New
England Biolabs cat#: B7703S) for at least one hour and the samples were analyzed by
Western blot according to standard protocols. Input lanes contained 1 microliter of cell
extract. Antibodies used to probe Western blots were either commercial (anti-myc from
Santa Cruz (9E10), anti-HA from Roche (12CA5)), from this lab (anti-LexA and antiMcm3), or were generously provided by other labs (anti-actin from A. Bretscher, antiStu2 from T. Huffaker, anti-Sir3 from R. Kamakaka).
Fluorescence-Activated Cell Sorting (FACS)
For FACS analysis, 1mL aliquots of growing yeast cells were spun down, and
fixed using cold 70% EtOH. The cells were then rinsed twice with 1mL of 50mM
NaCitrate. The cells were then sonicated briefly (3 times for 5 seconds) at setting 4 on
the VirSonic Ultrasonic Cell Disrupter 100 (SP Industries). 12 microliters of 10mg/mL
RNAse A (QIAGEN cat#: 1007885) was added and the cells were incubated at 42°C for
1 hour. 0.5mg of Proteinase K was added and the sample was incubated for 1 hour at
42°C. 1 microliter of 1mM SYTOX Green (Invitrogen Molecular Probes cat#: S7020)
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was added for each 1mL of cell suspension before processing the samples. The analysis
was performed at The Biomedical Sciences Flow Cytometry Core Laboratory at Cornell
University.
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RESULTS
Pre-RC components play a role in heterochromatic silencing.
Several previously identified pre-RC mutants were tested for silencing defects.
Interestingly, almost all replication mutants tested exhibited some level of silencing
defect both at the telomere (Figure 1A) as well as at the HMR (Figure 1B). This
observation raised the possibility that some pre-RC proteins may interact with silencing
factors. Indeed, when tested in a two-hybrid system using LexA binding domain (BTM)
and Gal4 activation domain (GAD) fusion proteins, several bait BTM-MCM protein
constructs showed an interaction with the prey GAD-SIR2 (Figure 1C), but not with the
empty GAD vector. The most obvious SIR2-interactor in this experiment was MCM7,
whereas MCM3, MCM5 and MCM6 constructs showed weaker interaction signals. In
addition, Mcm10 has been previously shown to interact with Sir2 (DOUGLAS et al. 2005;
LIACHKO and TYE 2005). Plasmids expressing mutant versions of Mcm10 (mcm10-43),
Mcm3 (mcm3-10), and Mcm7 (mcm7-1) failed to activate the LacZ reporter indicating
that their interaction with GAD-SIR2 was abolished (Figure 1D). These findings are
interesting because they suggest a greater interaction between silencing and replication
than previously described.
Mcm10 and Mcm3 interact with Sir2 in G2 phase in a DNA-independent manner.
Mcm10, Mcm3, and Mcm7 are all DNA replication proteins. Therefore, it is
important to examine whether their interaction with Sir2 is restricted within the context
of DNA replication. Since the process of DNA replication is limited to S phase, this
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question can be addressed by performing co-immunoprecipitation (Co-IP) experiments
on cells arrested in the G2/M phase of the cell cycle. Yeast cells expressing 3HA-tagged
Sir2, and 13myc-tagged Mcm10, or Mcm10-43 proteins were arrested at the beginning of
M phase using nocodazole, a microtubule inhibitor. Their FACS profiles showed strong
arrest phenotypes (Figure 2A). These arrested cultures were subsequently used for CoIPs. Immunoprecipitation either without anti-HA antibody or using an untagged Sir2
strain (data not shown) did not precipitate any assayed proteins. However, both Mcm3
and 13myc-Mcm10 were precipitated by the anti-HA antibody in a SIR2-3HA cell
extract. In all cases, Sir2-3HA failed to pull down either actin, or a microtubule
associated protein Stu2. There was no detectable change in the Mcm3/Sir2 interaction or
in the Mcm10/Sir2 interaction between asynchronous and G2/M arrested cells (Figure
2A). In both cases, Mcm10-43 mutant protein failed to interact with Sir2 and inhibited
the ability of Mcm3 to interact with Sir2 as well. In order to test whether the Sir2/Mcm
interaction is DNA dependent, we performed Co-IPs using extracts that were treated with
DNAseI (Figure 2B). Our results showed no difference in interaction between DNAseI
treated and untreated samples. In order to exclude the possibility that the Sir2-MCM
interaction is protected by silent chromatin, we performed a similar experiment in a sir3Δ
background strain and observed no effect of DNAse on the interaction between Mcm10
and Sir2 (Figure 2C). These findings correlate with the two-hybrid results and support
the hypothesis that DNA replication proteins, in this case Mcm10 and Mcm3, play a role
in silencing that may not be restricted to S-phase.
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A 53 amino acid domain in the C-terminus of Mcm10 is necessary and sufficient for
the interaction of Mcm10 with Sir2, Mcm3, and Mcm7.
The Mcm10-43 and Mcm10-1 mutant proteins are temperature labile proteins
(RICKE and BIELINSKY 2004; SAWYER et al. 2004) each containing a single amino acid
change that affects the overall structure of Mcm10 and destroys its interaction with Sir2.
Previous work showed that Mcm10 interacts with Sir2 and that this interaction depends
on the C-terminus of Mcm10 (DOUGLAS et al. 2005; LIACHKO and TYE 2005). To isolate
the domain that is responsible for the Mcm10/Sir2 interaction, bait plasmids expressing
fragments of the C-terminus of MCM10 were co-expressed with a SIR2 prey construct in
a yeast two-hybrid system. The BTM-MCM10 truncation constructs were designed by
using a comparative genomic approach to identify conserved regions within the Cterminus (data not shown). All BTM constructs, except one, expressed robustly in vivo as
shown by Western blots (Figure 3B). Several BTM-MCM10 truncation constructs
interacted with GAD-SIR2, but some did not (Figure 3A). The pattern of interactions
indicated that a region between amino acids Ser503 and Lys555 of Mcm10 was necessary
for the interaction with Sir2. The expression of the bait plasmid bearing this 53 amino
acid fragment resulted in an interaction with GAD-SIR2 in the two-hybrid system (Figure
3A). None of the truncation constructs activated the two-hybrid reporter when coexpressed with an empty GAD plasmid, suggesting that amino acids 503-555 of Mcm10
are necessary and sufficient for the interaction with Sir2.
To further characterize the Sir2-interaction domain of Mcm10 we used a
computational approach to identify potential secondary structures within this region.
Secondary structure prediction (http://www.compbio.dundee.ac.uk/~www-jpred/)
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suggested that there was an amphipathic helical region between amino acids Thr515 and
Tyr523 of Mcm10 (data not shown). To test whether this region was necessary for the
Sir2/Mcm10 interaction, site-directed mutagenesis was used to introduce one of three
mutations (T515V, I517T, and D519N) into this helical domain. Two of these mutations
(I517T and D519N) abolished the interaction between Mcm10 and Sir2 in two hybrid
assays (Figure 3C). In addition, deletion of the last 68 amino acids of Mcm10 (mcm10(1502)) also disrupted the Mcm10/Sir2 interaction. These mutations did not cause the
destabilization of the bait proteins (Figure 3D), suggesting that the loss of interaction was
due to the effect of the mutations on the structure of Mcm10. In addition, The D519N
mutant, as well as the truncation removing the interacting domain (1-502) abolished the
interaction of BTM-MCM10 with GAD-MCM3 and GAD-MCM7 (Figure 3C).
Mcm10 mediates the interaction between MCM2-7 and SIR2.
The disruption of Mcm10’s interaction with Sir2 as well as with Mcm3 and
Mcm7 by the mcm10-D519N mutation (Figure 3C) suggests that Mcm10 may mediate
the interactions between the MCM2-7 complex and Sir2. This possibility is further
supported by our Co-IP results which showed that the Sir2/Mcm3 interaction is disrupted
in mcm10-43 background (Figure 2A). To test this hypothesis, interaction studies were
performed using strains bearing different mcm mutant alleles. In a strain bearing the
mcm10-43 allele or the mcm10(1-502) allele, Sir2 was not able to efficiently coprecipitate Mcm10 nor Mcm3 (Figure 4A). However, some Sir2/Mcm3 interaction
remained in mcm10(1-502) background, suggesting that mcm10-43 has a greater effect on
the Sir/MCM2-7 interactions than mcm10(1-502). These Co-IP results support the two-
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hybrid data showing that mutant Mcm10 proteins are not able to interact with Sir2
(DOUGLAS et al. 2005; LIACHKO and TYE 2005). To test whether Mcm10 is also required
for the Sir2/Mcm7 interaction, yeast two-hybrid experiments were performed in a
mcm10-1 strain background. While BTM-MCM7 is able to interact with GAD-SIR2 in a
wild type strain background, this interaction disappears in a mcm10 mutant strain (Figure
4B). This finding supports the hypothesis that Mcm10 mediates the interaction between
MCMs and Sir2.
While Mcm10 is required for the interaction of Mcm3 and Mcm7 with Sir2, it is
also possible that Mcm3 and Mcm7 are required for the interaction of Mcm10 with Sir2.
To test this possibility, Co-IPs were performed in strains bearing mutant mcm3 or mcm7
alleles (Figure 4C). Both mcm3-10 and mcm7-1 mutations abolished the interaction
between Mcm3 and Sir2 suggesting that Mcm3 and Mcm7 may interact with Sir2 as a
complex. However, these mutations had no effect on the Mcm10/Sir2 interaction
supporting the hypothesis that Mcm10 acts as a bridge between the MCMs and the SIRs.
Mutations in the Sir2 interacting domain of Mcm10 cause defects in silencing, but
not replication.
To test what effect the mcm10 C-terminal mutations have on silencing and
replication, these mutations were introduced into the genome of a strain bearing a
telomeric silencing reporter. Both T515V and I517T mutations did not confer a
measurable silencing defect, while D519N conferred a slight defect which could be
complemented by a wild type copy of MCM10 (Figure 5A). This defect was relatively
weak compared to the defect conferred by the temperature sensitive mcm10-1 or mcm10-
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43 alleles. In addition, an mcm10(1-502) mutant strain bearing a deletion of the Cterminal 68 amino acids of MCM10 was viable, which is consistent with previous results
showing that this region is not essential for growth (DOUGLAS et al. 2005). This
truncation allele conferred a slight silencing defect (Figure 5A). This defect was very
similar to that caused by the mcm10-D519N allele, suggesting that the D519N mutation
has a significant effect on the structure of the C-terminus of Mcm10 since its phenotype
resembles a deletion of the C-terminus. Deletion of the C-terminal fragment in a mcm1043 background increased the silencing defect indicating that mcm10-43 retains some
silencing function that is mediated by the C-terminal domain.
Expression plasmids bearing different MCM10 alleles were transformed into a
silencing reporter strain bearing the temperature sensitive mcm10-1 mutation (Figure 5B).
Plasmids expressing MCM10, mcm10-T515V, and mcm10-I517T fully complemented
both the temperature sensitivity of mcm10-1 as well as its silencing defect. Plasmids
expressing mcm10-D519N and mcm10(1-502) complemented the temperature sensitivity,
but did not fully complement the silencing defect. These results further corroborate the
silencing phenotypes observed in Figure 5A. Expression of the mcm10(503-555) domain
did not complement the silencing defect nor the temperature sensitivity of the mcm10-1
strain. This suggests that although this short domain of Mcm10 is necessary and
sufficient for the interaction of Mcm10 with Sir2, it is not sufficient to restore silencing
or replication functions of Mcm10.
We have previously shown that second site suppressors of the conditional
lethality of mcm10-1 do not suppress the silencing defect caused by this mutation
(LIACHKO and TYE 2005). This phenotype-specific suppression suggests that the
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replication function of Mcm10 can be modulated independently of its silencing function.
Since the C-terminal mutations in Mcm10 cause silencing defects, we assayed them for
replication defects as well. To test the effect of C-terminal Mcm10 mutations on DNA
replication, minichromosome maintenance assays were performed. These assays are used
to measure the loss of an ARS-bearing plasmid, indicative of a defect in replication
(DONATO et al. 2006; MAINE et al. 1984). Despite the fact that the mutation in mcm10D519N caused a silencing defect, it did not cause a measurable minichromosome
maintenance defect (Figure 5C). Furthermore, deletion of the last 68 amino acids from
the C-terminus of Mcm10 in either wild type or mcm10-43 backgrounds did not increase
minichromosome loss (Figure 5C). These findings suggest that the replication function
of MCM10 is separate from its silencing function.
Mcm10 does not regulate the association of Sirs with silent chromatin.
Previous work has shown that Mcm10 plays a role in the maintenance of silent
heterochromatin, however very little is known about the mechanism through which this
occurs (LIACHKO and TYE 2005). One possibility is that Mcm10 may have an effect on
the association of Sir proteins with chromatin. A defect in such a function could lead to a
gradual dissociation of the Sirs from the silent regions without necessarily affecting the
initial establishment of silencing. To test this possibility, chromatin immunoprecipitation
(ChIP) experiments were conducted to measure the association of Sir2 with silenced
regions of the genome in mcm mutant strains (Figure 6). ChIP experiments were
performed on SIR2 (untagged) and SIR2-3HA strains using the anti-HA antibody and the
precipitated DNA was analyzed by real-time PCR.
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Our results show that Sir2-3HA readily associates with silent regions HMR-E and
HML-E, but not with a control gene region GPX1. No DNA was precipitated from a
strain bearing an untagged SIR2 allele. A control strain bearing the deletion of SIR4
abolished the interaction of Sir2-3HA with chromatin as previously shown (Figure 6A)
(RUSCHE et al. 2002). We have observed previously that mcm10-1 and mcm10-43
mutations cause the derepression of the HMR and HML loci (LIACHKO and TYE 2005).
Strains bearing mcm3-10 and mcm7-1 alleles also show similar defects in silencing
(Figure 1). However, neither mcm10-43 nor mcm3-10 strains had a measurable effect on
the association of Sir2 with these regions. We have also tested the effect of mcm alleles
on the association of Sir2 with silencing reporters used in Figure 1. We did not detect a
significant difference in Sir2’s association with the telomeric URA3 reporter nor the
hmr::ADE2 reporter (Figure 6B). These results suggest that Mcm10 does not regulate the
association of Sir2 with silent chromatin.
In addition, neither mcm10 nor mcm3 mutations affected the association of Sir2
with a1 or α2 genes (Figure 6). These genes reside within the HM loci and are silenced
by the spreading of the Sir2-4 proteins from the HMR and HML silencers (RUSCHE et al.
2002; RUSCHE et al. 2003). Our finding suggests that mcm mutations do not affect the
spreading of Sir proteins after the initial binding to the HMR and HML silencers.
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DISCUSSION
We have used several assays to demonstrate novel protein-protein interactions
between components of the replication fork complex (Mcm3 and Mcm7) and the Sir2
histone deacetylase is essential for silencing (Figure 1C). This study is the first direct
evidence that members of the replicative helicase physically interact with the chromatin
silencing machinery. This interaction is not affected by DNAse treatment, and persists in
the G2/M phases of the cell cycle (Figure 2), suggesting the possibility that it takes place
outside of the context of DNA replication. The mutant allele mcm7-1 disrupts the
interaction between Sir2 and Mcm3 (Figure 4C), but not between Sir2 and Mcm10. This
finding suggests that Mcm3 and Mcm7 act in a complex, possibly in a fashion similar to
their action in DNA replication. In addition, silencing assays performed on mutants of
these genes showed defects in telomeric as well as HMR silencing (Figure 1A and B).
Together these results implicate members of the pre-RC in transcriptional silencing. It is
not yet known by what mechanism these proteins influence the formation of silent
chromatin, but it has become clear that Mcm10 mediates these interactions. The net
outcome of a failure to mediate these interactions is that the silencing machinery that is
recruited to chromatin no longer efficiently silences chromatin.
Previously, Mcm10, an essential protein involved in both the initiation and
elongation of DNA replication (KAWASAKI et al. 2000; MERCHANT et al. 1997; RICKE
and BIELINSKY 2004) has been implicated in the maintenance of silencing (DOUGLAS et
al. 2005; LIACHKO and TYE 2005). Here we show that this protein interacts with several
members of the silencing machinery and is required for the interaction between Sir2 and
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Mcm3 and Mcm7 (Figure 2A, Figure 4). Careful dissection of this interaction has
isolated a short region at the C-terminus of the Mcm10 protein that is both necessary and
sufficient for the interaction with Sir2 (Figure 3A). Mutations in this region abolish not
only the interaction of Mcm10 with Sir2, but also with several previously characterized
interacting partners of Mcm10 involved in DNA replication (Figure 3C). However, only
the mutants that abolish interactions between Mcm10 as well as the other replication
factors are able to confer a silencing defect (Figure 3C, Figure 5). This observation
suggests that the function of Mcm10 in silencing may be as a mediator between these
other factors.
It is also notable that C-terminal mcm10 mutations confer much weaker silencing
defects than mcm10-1 and mcm10-43 mutations despite the fact that both types of
mutations disrupt interactions with Sir2. In addition, mutating the C-terminus of mcm1043 increases its already significant silencing defect (Figure 5A). One explanation is that
the C-terminus is only partially responsible for mediating these interactions and other
parts of Mcm10 also contribute. This idea is supported by the observation that in Co-IP
experiments C-terminal mutants disrupt the Sir2/Mcm10 interaction, but retain some of
the Sir2/Mcm3 interaction (Figure 4A). It is unlikely that an overall conformational
change of Mcm10-43p is the whole explanation for the disruption of Mcm10’s
interactions with Sir2 and Mcm3 (Figure 1) because a mutation in the C-terminus
exacerbated this phenotype (Figure 5A). A more plausible explanation for these
observations is that Mcm10’s function in silencing is more complex, possibly involving
numerous interactions with yet unidentified proteins. The C-terminal domain could
regulate some aspects of this function, whereas other aspects could be regulated by
21
another part of the protein, so mutating both regions of Mcm10 has a cumulative effect
on silencing. Recent studies suggest that Mcm10 may function as a ring complex of six
subunits (COOK et al. 2003; OKOROKOV et al. 2007). It is conceivable that individual
subunits of the Mcm10 complex may interact with a different set of interactors.
Several models have been proposed for the mechanism of Mcm10 function in
silent chromatin structure based on previous findings (LIACHKO and TYE 2005). One
model suggested that Mcm10 may be part of silent chromatin itself. However, a recent
study showed that Mcm10 is only associated with chromatin during S phase (RICKE and
BIELINSKY 2004), making this model unlikely since silent chromatin must persist
throughout most of the cell cycle. Another model suggested that Mcm10 may regulate
the association of Sir2 with chromatin. However results of this study have shown that
this is not the case (Figure 6). Another hypothesis suggested that Mcm10 regulates the
transition from initial binding of Sir proteins to their spreading along the chromatin. We
have shown that mcm mutations did not have an effect on the association of Sir2 with
HMR-E and HML-E silencers, nor with mating type genes a1 and α2 (Figure 6A), which
are silenced by the spreading of Sir proteins (RUSCHE et al. 2002; RUSCHE et al. 2003).
Neither did these mutations affect the chromatin association of Sir2 with telomeric or
HMR-based reporter genes which are also silenced by the spreading of the Sirs and silent
chromatin (Figure 6B). Since spreading of the Sirs requires the Sir2 deacetylase activity
(RUSCHE et al. 2002), this result also rules out Mcm10 playing a role in the activation of
Sir2. While it may seem counterintuitive that silencing can be disrupted without
affecting Sir association, these findings are consistent with a recent study showing that
22
the association of Sir proteins with silent regions are uncoupled from transcriptional
silencing at these regions (KIRCHMAIER and RINE 2006).
The findings that Mcm10 interacts with chromatin only during S phase, but can
interact with Sir2 in other phases of the cell cycle imply that the Mcm10/Sir2 interaction
occurs away from the chromatin. A consistent model is that Mcm10 stabilizes the
complex formation between Sir2 and additional factors that modify Sir2 in such a way to
make it more competent for silencing (Figure 7). If Mcm10 or other MCMs are
defective, unmodified or improperly associated, then Sir2 will be incorporated into
heterochromatin and silencing will be reduced. One potential player in such a
mechanism may be the Cdc7-Dbf4 kinase which has also been implicated in silencing
(AXELROD and RINE 1991; REHMAN et al. 2006). In S. pombe the homolog of Cdc7-Dbf4
has been shown to phosphorylate the homolog of HP-1 in a DNA replication-independent
manner (BAILIS et al. 2003). Cdc7-Dbf4 is also able to phosphorylate the Mcm2-7
complex, in a Mcm10-dependent manner (LEE et al. 2003). Since Mcm10 has been
shown to physically interact with both HP-1 and Mcm proteins (CHRISTENSEN and TYE
2003; MERCHANT et al. 1997), it is conceivable that in budding yeast Mcm10 is required
for Cdc7-Dbf4 to phosphorylate one of the SIRs, in lieu of HP-1, which is not found in
yeast. This model is also consistent with data showing that Mcm10 is required to
maintain an interaction between Cdc17 and Pol12, and that maintenance of this complex
is necessary for Pol12 phosphorylation (RICKE and BIELINSKY 2006). This idea is also
consistent with what is already known about the function of Mcm10 as a stabilizing
factor for larger complexes, such as the pre-RC and the Polymerase-α/primase complex.
23
This study raises interesting possibilities on the nature of the relationship between
silencing and replication. Past studies have implicated DNA replication factors in
connection with transcriptional silencing, however several other studies have shown that
the process of replication is not required for silencing (KIRCHMAIER and RINE 2001; LAU
et al. 2002; LI et al. 2001; MARTINS-TAYLOR et al. 2004; MILLER and NASMYTH 1984).
This apparent contradiction suggests that DNA replication factors may have nonreplication functions. Indeed, this has been clearly demonstrated with the ORC (BELL et
al. 1993; DILLIN and RINE 1997; EHRENHOFER-MURRAY et al. 1995; FOSS et al. 1993;
FOX et al. 1997; HOU et al. 2005; HSU et al. 2005; LOO et al. 1995; MICKLEM et al. 1993;
TRIOLO and STERNGLANZ 1996; ZHANG et al. 2002). In addition, recent work has shown
a genetic interaction between Sir2 and members of the pre-RC where deletion of SIR2
rescues temperature-sensitive pre-RC mutants (CRAMPTON et al. 2008; PAPPAS et al.
2004). This finding suggests that Sir2 plays a role in regulating replication. It is not yet
known whether the interactions shown in this study are relevant for both replication and
silencing. Notably, second site suppressors of mcm10-1 fail to rescue its silencing defect
(LIACHKO and TYE 2005). Also, several silencing defective mcm10 mutants do not
exhibit replication defects (Figure 5). These observations, along with the finding that the
Mcm-Sir2 interaction persists outside of S-phase (Figure 2A) argue that Mcm10’s
silencing function may be separate from its replication function. Future studies in this
area should further elucidate the connection between DNA replication and silent
chromatin.
24
ACKNOWLEDGEMENTS
We would like to thank the Bretscher, Huffaker, Roberts and Kamakaka labs for
providing antibodies and strains. We also thank Amy Lyndaker for help with ChIP and
Justin Donato and Tim Christensen for helpful discussions and critical reading of the
manuscript. This project is supported by NSF-MCB0453773 and NIH GM072557. IL is
supported by NIH training grant 3 T32 GM07617-25S2.
25
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29
Table 1 – Strains Used in this Study
Strains
Isogenic to W303
W303-1A
W303-1B
BTY100
BTY101
BTY103
BTY102
ILY115
ILY360
ILY171
ILY180
ILY270
ILY248
ILY288
ILY295
ILY298
ILY332
ILY330
ILY331
ILY336
ILY334
ILY178
ILY253
ILY255
ILY254
ILY264
ILY230
ILY232
ROY1515
ILY273
ILY274
ILY275
ILY276
ILY185
ILY338
ILY346
ILY348
ILY349
ILY351
ILY353
ILY355
ILY357
ILY328
Other
Backgrounds
EGY40[pSH18-34]
MATa ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3-1
MATα ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3-1
W303 MATa mcm10-1
W303 MATα mcm10-1
W303 MATa mcm10-43
W303 MATα mcm10-43
W303 MATa mcm7-1(cdc47-1)
W303 MATα mcm3-10
W303 MATa hmr::ADE2 adh4::URA3 Tel (VII-L)
ILY171 mcm10-1
ILY171 mcm10-43
ILY171 MATα 13myc-MCM10 TRP1
ILY248 MATa mcm10-T515V
ILY248 MATa mcm10-I517T
ILY248 MATa mcm10-D519N
ILY248 MATa mcm10-43
ILY273 mcm10(503-571)::HIS3
ILY275 mcm10-43(503-571)::HIS3
ILY330 MATα hmr::ADE2 adh4::URA3 Tel (VII-L)
ILY331 hmr::ADE2 adh4::URA3 Tel (VII-L)
ILY171 mcm2-1
ILY171 mcm3-10
ILY171 cdc54-1
ILY171 MATα mcm7-1
ILY171 cdc6-3
MATa 13myc-MCM10 TRP1
MATa 13myc-mcm10-43 TRP1
Source
ILY230 6xHis-3xHA-SIR2
ILY230 MATα 6xHis-3xHA-SIR2
ILY232 6xHis-3xHA-SIR2
ILY232 MATα 6xHis-3xHA-SIR2
W303 MATa sir2::HIS3
ILY273 mcm3-10
ILY273 mcm7-1
ILY273 sir4::HIS3
ILY171 6xHis-3xHA-SIR2
ILY180 6xHis-3xHA-SIR2
ILY332 6xHis-3xHA-SIR2
ILY253 MATα 6xHis-3xHA-SIR2
ILY254 6xHis-3xHA-SIR2
ILY273 sir3:HIS3
R. Rothstein
R. Rothstein
This Lab
This Lab
This Lab
This Lab
This Lab
This Lab
This Lab
This Lab
This Lab
This Study
This Study
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This Study
This Study
This Study
This Study
This Study
This Study
This Study
This Study
This Study
This Study
This Study
This Study
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R. Kamakaka
This Study
This Study
This Study
This Study
This Lab
This Study
This Study
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This Study
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This Study
MATa ura3-52 trp1-1 leu2-3,112 [pSH18-34]
E. Golemis
W303 MATa 9xMyc-NET::LEU2 pep::4Δ::TRP1 ade2 LYS2 6xHis-3xHA-SIR2
30
Table 2 – Plasmids Used in this Study
Plasmid Name
pRS315
pRS315MCM10
pGAD2F
pBTM116
pSH18-34
pGADgw
pBTMgw
pGBKgw
pBTMMCM10
pBTMmcm10-1
pBTMmcm10-43
pBTMMCM10(386-end)
pBTMMCM10(480-end)
pBTMMCM10(503-end)
pBTMMCM10(143-555)
pBTMMCM10(386-555)
pBTMMCM10(386-512)
pBTMMCM10(386-480)
pBTMMCM10(503-555)
pBTMMCM10(503-555)T515V
pBTMMCM10(503-555)I517T
pBTMMCM10(503-555)D519N
pBTMMCM10-T515V
pBTMMCM10-I517T
pBTMMCM10-D519N
pBTMMCM2
pBTMMCM3
pBTMMCM4
pBTMMCM5
pBTMMCM6
pBTMMCM7
pBTMCDC6
pBTMCDC45
pGADSIR2
pGADMCM7
YCp1
Description
YCP LEU2
YCP LEU2 MCM10
2μ LEU2 GAD4-AD
2μ TRP1 LEXA-DBD
URA3 LacZ with LEXA binding sites
pGAD2F with Gateway Cassette
pBTM116 with Gateway Cassette
pGBKT7 with Gateway Cassette
r
amp
pBTMgw MCM10
pBTMgw mcm10-1
pBTMgw mcm10-43
pBTMgw MCM10 (386-571)
pBTMgw MCM10 (480-571)
pBTMgw MCM10 (503-571)
pBTMgw MCM10 (143-555)
pBTMgw MCM10 (386-555)
pBTMgw MCM10 (386-512)
pBTMgw MCM10 (386-480)
pBTMgw MCM10 (503-555)
Source
New Engalnd
Biolabs
This Lab
S. Fields
S. Fields
S. Fields
This Study
This Study
This Study
This Study
This Study
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This Study
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This Study
This Study
This Study
This Study
pBTMgw MCM10 (503-555)-T515V
This Study
pBTMgw MCM10 (503-555)-I517T
This Study
pBTMgw MCM10 (503-555)-D519N
pBTMgw MCM10-T515V
pBTMgw MCM10-I517T
pBTMgw MCM10-D519N
pBTMgw MCM2
pBTMgw MCM3
pBTMgw MCM4
pBTMgw MCM5
pBTMgw MCM6
pBTMgw MCM7
pBTMgw CDC6
pBTMgw CDC45
pGADgw SIR2
pGADgw MCM7
LEU2 CENV ARS1
This Study
This Study
This Study
This Study
This Lab
This Lab
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This Lab
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31
FIGURE LEGENDS
Figure 1 – Pre-RC proteins play a role in silencing. (A) Silencing reporter strains
bearing conditional alleles of pre-RC proteins were plated at 30 degrees Celsius on media
containing or lacking 5-FOA, a chemical which kills cells expressing URA3. Strains used
were ILY171 (WT), ILY270 (mcm10-43), ILY178 (mcm2-1), ILY253 (mcm3-10),
ILY255 (mcm4-1, or cdc54-1), ILY254 (mcm7-1), and ILY264 (cdc6-3). Silencing
defects are indicated by lack of growth on 5-FOA media. Several of the mutant strains
show telomeric silencing defects. (B) The effect of mcm mutants on HMR silencing was
assayed using strains bearing an hmr::ADE2 reporter (ILY171, ILY270, ILY253,
ILY254). When the HMR locus is silenced, hmr::ADE2 cells form pink colonies, the
white color of the mutant strain indicates a derepression of the HMR locus (left panel).
No effect was observed on the color of control mutant strains BTY103, ILY115, and
ILY360 (right panel). (C) Yeast two-hybrid experiments were conducted using BTM bait
constructs containing pre-RC genes and prey constructs expressing either GAD-SIR2 or
GAD (empty vector). MCM3 and MCM7 constructs showed strong activation of the LacZ
reporter (indicated by the blue color) with GAD-SIR2 while MCM5 and MCM6 showed
weaker activation. (D) Bait plasmids expressing pre-RC mutants fail to interact with
GAD-SIR2 as well as the wildtype fusion proteins.
Figure 2 – Sir2’s interaction with Mcm3 and Mcm10 is not dependent on DNA and can
occur in G2/M phase. (A) Strains ILY273 (SIR2-HA) and ILY275 (SIR2-HA mcm10-43)
were arrested using nocodazole. FACS analysis shows the arrest profiles of cells used for
32
Co-IP experiments. Both arrested and asynchronous cultures were used for Co-IPs with
either no antibody added or using anti-HA antibody (anti−HA IP). Precipitated samples
were analyzed by Western blots probed for myc-Mcm10, Mcm3, actin, and Stu2. 1μL of
cell extract was used as input control (IN) for each immunoprecipitated sample (IP). (B)
Co-IPs were performed using strain ILY273. Before the addition of antibody, cell
extracts were either treated with DNAseI (two rightmost lanes) or not (four leftmost
lanes). (C) The same as in (B), only using strains ILY230 (two leftmost lanes) and
ILY328 (four rightmost lanes).
Figure 3 – Amino acids Ser503-Lys555 of Mcm10 are necessary and sufficient for
interaction with Sir2. (A) Yeast two-hybrid experiments were performed using the
EGY40 strain and BTM bait constructs expressing truncations of MCM10 as indicated.
The blue color indicates the activation of the LacZ reporter signaling a positive
interaction. (B) Western blots were used to confirm the expression of the truncation
constructs used in (A). (C) Constructs expressing mutant versions of MCM10 were used
in two-hybrid experiments. (D) The expression of bait constructs from (C) assayed by
Western blots.
Figure 4 – Mcm10 mediates Sir2’s interaction with Mcm3 and Mcm7. (A) Co-IP
experiments were performed on strains ILY273 (first four lanes), ILY275 (fifth and sixth
lanes), and ILY330 (the two rightmost lanes). The Western blot was probed with antimyc and anti-Mcm3 antibodies. (B) The two-hybrid reporter plasmid pSH18-34 was
transformed into strains W303-1A (WT), BTY100 (mcm10-1), and ILY185 (sir2Δ).
33
Two-hybrid bait and prey constructs bearing SIR2, MCM3, and MCM7 were used to
assay interactions. (C) Co-IP experiments were performed in strains ILY273 (first four
lanes), ILY338 (mcm3-10), and ILY346 (mcm7-1) as in (A) and (B).
Figure 5 – C-terminal mutants of MCM10 exhibit silencing, but not replication defects.
(A) Serial dilutions of mcm10 mutant strains bearing a telomeric URA3 reporter gene
were plated on media with or without 5-FOA to assay telomeric silencing. The strains
used were ILY248 (WT), ILY180 (mcm10-1), ILY288 (mcm10-T515V), ILY295
(mcm10-I517T), ILY298 (mcm10-D519N), ILY336 (mcm10(1-502)), ILY332 (mcm1043), and ILY334 (mcm10-43(1-502)). The mcm10-D519N mutant strain displays a
silencing defect, but the same strain with a YCp plasmid bearing a copy of MCM10
(pRS315-MCM10) does not. (B) mcm10-1 mutant strain ILY180 was transformed with
two-hybrid bait plasmids expressing MCM10 alleles as indicated. The cells were assayed
for silencing activity on 5-FOA media and for temperature sensitivity on complete media
at 37°C. (C) Minichromosome maintenance assays performed on strains from (A) to
measure the rate of loss of ARS1-bearing plasmid YCp1.
Figure 6 – Mcm10 does not regulate the association of Sir2 with chromatin. (A) DNA
obtained by anti-HA ChIP from strains ILY230 (untagged), ILY273 (WT), ILY275
(mcm10-43), ILY253 (mcm3-10), and ILY348 (sir4Δ) was analyzed by quantitative realtime PCR. The bars show relative enrichment of IP samples over 10% input controls.
The sites probed were the silencers HMR-E, HML-E, the nearby genes a1 and α2, and the
GPX1 gene region as a negative control. (B) A similar ChIP experiment using strains
34
bearing telVII::URA3 and hmr::ADE2 silencing reporters. The mcm mutants did not
significantly reduce the amount of DNA precipitated with an anti-HA antibody. This
holds true for both the telomeric and HMR loci. The strains used were ILY171
(untagged), ILY351 (mcm10-1), ILY353 (mcm10-43), ILY355 (mcm3-10), ILY357
(mcm7-1). All ChIP experiments were performed at 30°C.
Figure 7 – Model for MCM function in silencing. (A) Mcm10 stabilizes the interactions
between Sir2 and Mcm2-7 proteins. This complex can be acted upon by a set of
modifying factors. The modified version of Sir2 is then assembled into functional silent
chromatin. (B) If MCM10 is mutated, it cannot stabilize the complex in (A) and therefore
fewer of the Sir2 molecules are modified, resulting in weaker silencing.
Supplementary Figure 1 – DNAse treatment of extracts used for Co-IP in Figure 2B.
Genomic DNA was purified from ILY273 cell extracts that was used for coimmunoprecipitation experiments. DNA was purified from freshly made cell extract
(untreated), or from the same extract after the sonication step (Sonicated). Fresh and
sonicated extracts were also treated with DNAse (+DNAse and Sonicated +DNAse
respectively) prior to the immunoprecipitation procedure.
Supplementary Figure 2 – Mutations in the mcm10(503-555) domain inhibit interaction
with Sir2. (A) Yeast two hybrid experiments were performed using bait constructs
expressing wild type and mutant versions of the mcm10(503-555). (B) Western blotting
shows that all three mutant constructs are expressed robustly.
35
36
Liachko_Figure 1
Telomere
URA3
Chr. VII
HMR::ADE2
WT
Growth
FOA
B
WT
mcm10-43
mcm3-10
mcm4-1
mcm7-1
mcm3-10
mcm2-1
mcm7-1
mcm3-10
mcm10-43
Control
WT
mcm10-43
A
cdc6-3
mcm7-1
2
R
SI
-
C
AD GAD
G
BTM
G
BTM-MCM10
BTM-MCM3
BTM-mcm10-43
BTM-MCM4
BTM-MCM3
BTM-MCM5
BTM-mcm3-10
BTM-MCM6
BTM-MCM7
BTM-MCM7
BTM-mcm7-1
BTM-CDC45
R
SI
-
AD
BTM-MCM2
BTM-CDC6
2
D
C
d
IN
IN
IP
IP
IP
IN
IN
IN
IP
IP
S
an IR2
+ ti- -H
D HA A
N
As IP
e
IN
a R2
- D nti- -H
N HA A s
As IP ir
3D
e
S
an IR2
+ ti- -H
D HA A
N
As IP sir3
D
e
IP
SI
B
S
N IR2
o
- D a -H
N ntib A
As o
e dy
S
an IR2
- D ti- -H
N HA A
As IP
e
IN
S
an IR2
- D ti- un
N HA ta
As IP gg
e
e
A
S
N IR2
Lo o a -H
g nti A
ph bo
as dy
e
SI
a R2
Lo nti- -H
g HA A
ph I
as P
e
SI
a R2
G nti- -H
2/ HA A
M
ph IP
as
SI e
a R2
Lo nti- -H
g HA A
ph I m
as P cm
10
e
S
-4
3
an IR2
G ti- H
2/ HA A
M
m
ph IP cm
as
10
e
-4
3
Liachko_Figure 2
IP
IN
IN
IN
IP
IN
IP
anti Mcm3
anti Myc (Mcm10)
anti actin
anti Stu2
IP
anti Mcm3
anti Myc (Mcm10)
IP
anti Myc (Mcm10)
C
BTM-mcm10-T515V
BTM-mcm10-I517T
BTM-mcm10-D519N
BTM-mcm10(1-502)
AD AD
G
1-571
386-571
480-571
2
AD GAD
G
R
SI
7
3
CM CM
-M -M
AD GAD
G
B
BTM
D
BTM-MCM10
BT MC
M M1
-m 0
BT cm
1
M
-m 0BT cm T51
5V
M
1
-m 0I
BT cm 51
7
M
-m 10- T
D
cm
5
10 19N
(1
-5
02
)
G
M
I
-S
M
BT -MC
M M
BT -mc 10
M m1 (1BT -mc 0 ( 571
M m1 38 )
BT -mc 0 ( 6-5
M m1 48 71)
BT -mc 0 ( 0-5
M m1 50 71)
BT -mc 0 ( 3-5
M m1 14 71)
BT -mc 0 ( 3-5
M m1 38 55)
BT -mc 0 ( 6-5
M m1 38 55)
-m 0 6cm (3 51
10 86- 2)
(5 48
03 0)
-5
55
)
A
BT
BTM-MCM10
BT
Liachko_Figure 3
R2
503-571
143-555
anti-LexA
386-555
386-512
386-480
503-555
anti-LexA
S
N IR2
o
an -HA
tib
od
y
SI
R
an 2
ti- -H
H A
A
IP
SI
m R
an cm 2-H
ti- 3- A
H 10
A
IP
SI
m R
an cm 2-H
ti- 7- A
H 1
A
IP
B
C
Background
A
S
M IR2
C
N M -H
o
an 10 A
tib -m
od yc
y
SI
R
M 2
an CM -H
ti- 10 A
H -m
A
IP yc
SI
m R2
an cm -H
ti- 10 A
H -4
A 3
IP -m
yc
SI
m R2
an cm -H
ti- 10 A
H (1
A -5
IP 0
2)
-m
yc
Liachko_Figure 4
IN
IN
IP
IP
IN
IP
BTM
GAD
IN
IN
IP
IN
IP
IP
IN
MCM7 SIR2
IN
IP
anti Mcm3
anti Myc (Mcm10)
MCM10 MCM7 MCM10
SIR2
WT
mcm10
sir2D
IP
anti Mcm3
anti Myc (Mcm10)
anti Stu2
anti actin
Telomere
URA3
FOA
Chr. VII
Liachko_Figure 5
Growth
WT
mcm10-1
mcm10-T515V
mcm10-I517T
A
mcm10-D519N
mcm10-D519N
+pRS315-MCM10
WT
mcm10 (1-502)
mcm10-43
mcm10-43 (1-502)
mcm10-D519N
FOA 30 C
CM 37 C
Growth
BTM
BTM-MCM10
BTM-mcm10-T515V
BTM-mcm10-I517T
BTM-mcm10-D519N
BTM-mcm10 (1-502)
BTM-mcm10 (503-555)
0.20
0.15
0.10
0.05
0.00
m
cm cm1
10 0-T 1
m
cm 51
m 10 5V
cm -I
10 517
T
m
cm D51
10 9N
(
m
m 1-5
cm cm 02
10 10 )
-4
3 43
(1
-5
02
)
T
W
m
C
Loss/generation
B
Liachko_Figure 6
A
Enrichment (arbitrary units)
0.10
untagged
WT
mcm10-43
mcm3-10
sir4D
0.08
0.06
0.04
0.02
0.00
B
HMR-E
HML-E
a1
a2
GPX1
telVIIL::URA3 hmr::ADE2
Enrichment (arbitrary units)
0.032
untagged
WT
mcm10-1
mcm10-43
mcm3-10
0.024
0.016
mcm7-1
0.008
0.000
URA3
ADE2
GPX1
Liachko_Figure 7
A
Mcm2-7
Modifying
Enzymes
Mcm10
Sir
Sir
Sir Sir Sir Sir Sir Ac Ac
More Silencing
B
Mcm2-7
Mcm10
Sir
Sir
Sir Sir Sir Sir Sir Ac Ac
Less Silencing