MCM proteins and DNA replication
Domenico Maiorano, Malik Lutzmann and Marcel Méchali
The MCM proteins identify a group of ten conserved factors
functioning in the replication of the genomes of archae and
eukaryotic organisms. Among these, MCM2–7 proteins are
related to each other and form a family of DNA helicases
implicated at the initiation step of DNA synthesis. Recently this
family expanded by the identification of two additional
members that appear to be present only in multicellular
organisms, MCM8 and MCM9. The function of MCM8 is
distinct from that of MCM2–7 proteins, while the function of
MCM9 is unknown. MCM1 and MCM10 are not related to this
family, nor to each other, but also function in DNA synthesis.
Addresses
Institute of Human Genetics, CNRS, 141, rue de la Cardonille,
34396 Montpellier, France
Corresponding author: Méchali, Marcel ([email protected])
regulation allows control of replication origin firing, which
is crucial to restrict the replication of the chromosome to
only one round per cell cycle. However, MCM2–7 are
widely distributed onto chromatin and not concentrated
at sites of DNA synthesis (replication foci), an issue that is
not consistent with their proposed function as helicases
also during the elongation stage of DNA synthesis.
MCM1 and MCM10 do not belong to this family,
although they are also conserved in higher eukaryotes.
MCM1 is a transcription factor that may play a role in
DNA synthesis, while MCM10 appears to be directly
required to initiate DNA synthesis.
Here we will review very recent advances on the understanding of MCM function in DNA synthesis and their
relevance in clinical application.
MCM1
Current Opinion in Cell Biology 2006, 18:130–136
This review comes from a themed issue on
Cell regulation
Edited by Claude Prigent and Bruno Goud
Available online 21st February 2006
0955-0674/$ – see front matter
# 2006 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.ceb.2006.02.006
Introduction
In eukaryotic cells initiation of DNA synthesis occurs at
defined sites (replication origins) scattered along the
chromosome. Replication origins are bound by the origin
recognition complex (ORC), which permits the recruitment of replication factors through a multistep reaction
leading to formation of replication forks, the functional
units of DNA synthesis [1,2]. MCM proteins were first
identified in the yeast Saccharomyces cerevisiae as mutants
defective in the maintenance of minichromosomes, suggesting a role in DNA replication. Six of them (MCM2–7)
are related to each other and interact to form a stable
heterohexamer in solution [3,4]. Extensive genetic and
biochemical characterization of MCM2–7 proteins support a role for these proteins at the initiation step of DNA
synthesis as DNA helicases that melt the DNA double
helix. They are recruited to DNA replication origins in a
highly regulated reaction at mitotic exit, leading to formation of the pre-replicative complex (pre-RC) [1].
MCM2–7 proteins bind to chromatin in a cell-cycledependent manner, being tightly bound in late mitosis
and G1 while being removed in S- and G2-phases [4]. This
Current Opinion in Cell Biology 2006, 18:130–136
MCM1 belongs to the MADS box transcription factor
family, which can interact with several cofactors to bind
their cognate DNA sequences cooperatively. Until
recently the role of MCM1 in DNA replication was
supposed to be rather indirect, since MCM1 regulates
the expression of CDC6 and some MCM2–7 genes [3]. A
more direct involvement of MCM1 in DNA replication
has recently been reported, showing that it binds to
multiple sites of yeast autonomously replicating
sequences (ARSs) [5], and that the activity of ARSs
can be stimulated by MCM1 binding [6]. It is now
important to determine how MCM1 stimulates ARS
activity, whether by recruiting replication factors and/or
by inducing chromatin remodelling.
The MCM2–9 protein family
Two additional members of the MCM2–7 family were
recently identified, MCM8 and MCM9 (Figure 1). On the
basis of sequence homology, these latter are absent in
yeast and nematodes. MCM9 exists only in vertebrates, as
it is absent in Drosophila [7], and it is more closely related
to MCM8 than to the other MCM2–7 proteins, suggesting
a recent duplication of the MCM8 gene (Figure 1a).
MCM9 is the most recently discovered MCM protein
and is likely to be the last MCM2-9 family member, as
judged by genomic data. The MCM9 N-terminus is
highly conserved amongst species, whereas the C-terminus is much less conserved (Figure 1b). The N-terminus
contains the MCM2–8 signature, including canonical
Walker A and B motifs similar to those in MCM8. Interestingly, MCM9 is by far the biggest MCM2–8-like
member owing to its extended C-terminal region, which
is unique to MCM9 and does not contain any known
motifs.
www.sciencedirect.com
MCM proteins and DNA replication Maiorano, Lutzmann and Méchali 131
Figure 1
The MCM2-9 super-family. (a) Phylogram of human MCM2-9 proteins (adapted from [7]) was calculated with ClustalW. (b) Alignment of human
MCM2-9 proteins. Bars represent the indicated proteins. The MCM2–7 family domain is shown in grey, and the region encompassing the
Walker A and B motif is shown in black. Numbers indicate amino acids.
MCM2–7
MCM2–7 proteins are recruited to replication origins by
two factors, Cdc6 and Cdt1. Although these latter can
bind to chromatin independently of each other, experiments with Xenopus egg extracts suggest that only a
sequential binding of Cdc6 followed by Cdt1 can lead
to the assembly of functional MCM2–7 complexes on
chromatin [8]. Moreover, in budding yeast, ATP hydrolysis mediated by a complex made of ORC and Cdc6
promotes efficient MCM2–7 loading [9,10]. These findings suggest that the functional chromatin assembly of
MCM2–7 requires initial binding of Cdc6 to ORC to form
a functional ORC–Cdc6 ATPase, while Cdt1 may link
this ATPase to the MCM2–7 complex (Figure 2). In
multicellular organisms, loading of MCM2–7 is also regulated by the geminin protein, which binds to Cdt1 onto
www.sciencedirect.com
chromatin [11,12] and thereby blocks the interaction of
Cdt1 with MCM2–7 and further MCM2–7 recruitment
onto chromatin after pre-RC formation [13,14]. In mammalian cells geminin may also contribute to pre-RC
formation by stabilizing Cdt1 in G2/M phases [15].
The mechanism by which MCM2–7 proteins function as
DNA helicases remains puzzling. Multiple complexes of
MCM2–7 proteins are assembled around an origin
(Figure 2), but it is currently unknown how many of
these complexes are subsequently activated to be functional. A model has also been proposed that places MCM
helicase at a fixed distance from replication forks [16].
Confusingly, the MCM2–7 heterohexamer has no DNA
helicase activity in vitro, while helicase activity is
observed with the MCM4/6/7 sub-complex in vitro
Current Opinion in Cell Biology 2006, 18:130–136
132 Cell regulation
Figure 2
Speculative model of MCM function. MCM1 may stimulate formation of pre-replicative complexes by recruiting either chromatin remodelling
complexes or replication factors such as ORC and/or unidentified factors. Following formation of the ORC-Cdc6 ATPase and Cdt1 binding, multiple
MCM2–7 complexes are loaded using ATP hydrolysis. MCM2–7 then recruits the Cdc7/Dbf4 kinase (DDK) and homocomplexes of MCM10. All
MCM2–7 complexes can potentially recruit DDK and MCM10 proteins. The RecQL4 helicase may also be recruited at this stage (not shown).
S-phase cyclin-dependent kinases (S-CDKs) are then recruited to origins by interaction with Cdc6 [50]. This step allows recruitment of pre-initiation
complex (preIC) components, such as Cdc45, Cut5, GINS, Sld2, Sld3 and, in multicellular organisms, the geminin protein binding to Cdt1.
Recruitment of S-CDKs by Cdc6 may allow the activation of only the MCM2–7 helicase (as double hexamer, shown in green) most proximal to Cdc6,
which might displace the inactive MCM2–7 complexes around the origin (shown in green). Unwinding stimulates binding of the single stranded DNA
binding protein RPA and the loading of DNA polymerase-a-primase through interaction with MCM10. Initiation of DNA synthesis promotes both
Cdt1 destruction and locking of the origin in the unlicensed state by oligomerization of geminin. Adaptors (Tof1, Csm3, Mrc1/Claspin and others)
linking MCM2–7 with preIC and DNA polymerases are also loaded at this stage. Formation of functional replication forks by loading of DNA
polymerase-d may permit binding of MCM8 and displacement of the remaining MCM2–7 complex from replicating chromatin (not shown).
[17]. In those Archaea organisms having only one MCM
protein, a homohexamer with DNA helicase activity can
be found, although its role in DNA synthesis has not yet
been addressed (see [18] and references therein). Some
evidence for DNA unwinding by MCM2–7 in eukaryotes
has been provided using a nucleus-free in vitro system
derived from Xenopus eggs. In this system, DNA unwinding is inhibited with neutralizing antibodies to MCM2–7
proteins or by inhibition of MCM7 activity with a fragCurrent Opinion in Cell Biology 2006, 18:130–136
ment of the retinoblastoma protein [19,20]. It is likely that
the helicase activity of MCM2–7 is stimulated in vivo by
Cdc45, a protein required to recruit DNA polymerase-a
onto chromatin. A complex between MCM2–7 and the
Cdc45 protein has been observed [21,22].
Although MCM2–7 proteins can be immunoprecipitated
from chromatin fragments containing proteins implicated
in the formation of replication forks [23], a physical
www.sciencedirect.com
MCM proteins and DNA replication Maiorano, Lutzmann and Méchali 133
interaction with replication fork core components, such as
DNA polymerases or the single stranded DNA binding
protein RPA, has not yet been found. Interestingly, in
whole cell extracts, an interaction between two proteins
involved in the checkpoint response, Tof1 and Mrc1, and
subunits of the MCM2–7 complex [21] has been detected
in budding yeast, during both normal replication and fork
arrest. Tof1 and Mrc1 co-localize with DNA polymerasea in yeast, as shown by Chip-on-Chip analysis [24],
suggesting that MCM2–7 may be linked to DNA polymerases by these and/or other proteins. This interaction
may be important to couple DNA synthesis to cell cycle
progression during replication stress or DNA damage
[25]. However, in Xenopus, an interaction between the
putative Mrc1 homolog (claspin) and MCM2–7 has not
been detected [22] and whether this interaction is conserved in mammalian cells is not yet determined.
MCM2–7 proteins are irreversibly released from chromatin during DNA synthesis. In the yeast Saccharomyces
cerevisiae, MCM2–7 are exported from the nucleus during
S phase. Recent findings suggest that this regulation
requires a newly identified nuclear export signal (NES)
in MCM3, which cooperates with S-CDKs activity to
exclude MCM2–7 proteins from the nucleus [26]. This
regulation appears to be specific to budding yeast, as in
the related yeast Schizosaccharomyces pombe, as well as in
higher eukaryotes, MCM2–7 are released free in the
nucleoplasm and are not exported [4]. In complex eukaryotes, Cdt1 destruction and geminin activity appear to be
critical elements suppressing MCM2–7 chromatin binding in S phase [27–31,32].
MCM8
MCM8 was identified in human cells as a gene targeted by
the human hepatitis B virus in patients with liver cancer
[33]. Preliminary characterization of MCM8 in Hela cells
has shown that it is not associated with MCM2–7 proteins
and that it binds chromatin after MCM2–7, suggesting
that it functions after pre-RC formation [33]. Characterization of a Xenopus MCM8 homologue has confirmed and
extended these findings [34]. Moreover, it was shown that
MCM8 associates with chromatin at the onset of S-phase.
Aphidicolin, an inhibitor of DNA polymerases that blocks
the elongation step of DNA synthesis but permits preRC
formation, prevents MCM8 chromatin loading in Xenopus
[34] and human cells [33], suggesting that MCM8 functions after formation of functional replication forks.
Removal of MCM8 from egg extracts slows down
DNA synthesis and results in accumulation of replication
intermediates, but does not affect pre-RC formation, such
as the chromatin assembly of both ORC and Cdc6 [33]
(Maiorano, Lutzmann and Méchali, unpublished).
ATPase and DNA helicase activity are found associated
with recombinant MCM8 in vitro, independently of the
MCM2–7 complex. MCM8 localizes to replication foci
together with the 34 kDa subunit of the RPA complex.
www.sciencedirect.com
Consistent with these findings, in the absence of MCM8
the binding of RPA34 and DNA polymerase-a to chromatin is deficient. Altogether, these data support a role for
MCM8 as a DNA helicase in unwinding during replication. As removal of MCM8 from egg extracts does not
completely inhibit DNA synthesis, other helicases may
compensate the absence of MCM8, such as MCM2–7,
MCM9 and/or the recently identified RecQL4 helicase,
which is essential to load RPA onto origins [35]. Alternatively, MCM8 may stimulate DNA synthesis and take over
the function of MCM2–7 in S-phase, while MCM2–7 are
excluded from chromatin by the passage of replication
forks. An independent study in human cells has suggested
that MCM8 binds to chromatin throughout the cell cycle
[36], in contrast to the observations in human cells [33] and
in Xenopus [34]. Down-regulation of MCM8 by siRNA in
cultured cells induces an S-phase delay, and results in low
levels of Cdc6 bound to chromatin, suggesting that MCM8
may be required to load Cdc6 onto DNA replication origins
to form pre-RCs. An alternative interpretation of this result
may be that MCM8 affects Cdc6 loading onto chromatin
after pre-RC formation. In Xenopus there is evidence that
Cdc6 is reloaded onto chromatin after initiation of DNA
synthesis [37]. Therefore, the lower levels of Cdc6
observed on chromatin following down-regulation of
MCM8 expression could be explained if Cdc6 reloading
is inhibited as a result of inhibition of the elongation step of
DNA synthesis. More work will be required to solve these
apparently conflicting issues in human cells.
MCM9
MCM9 was identified by searching for MCM2–8-like
proteins in the sequence databases [7,38]. A fragment
of the MCM9 gene has been reported [38,39] and the full
length genes and proteins have also been identified [7].
MCM9 is expressed in a variety of tissues in mouse and
humans. The role of this protein in DNA synthesis has
not yet been addressed.
MCM10
MCM10 is ubiquitous in eukaryotes and contains a
CCCH-type Zn-finger required for self-assembly into
homocomplexes which is essential for growth in budding
yeast [40]. MCM10 assembles onto origins of replication
after MCM2–7, apparently in two steps. It is first
recruited to origins of replication before CDC45, which
needs MCM10 to bind to chromatin [41]. MCM10 may
also be recruited after CDC45 in a complex with DNA
polymerase-a-primase [42]. In this conclusive work
[42], a fraction of S. cerevisiae MCM10 is shown to form
a soluble complex with DNA polymerase-a-primase.
Surprisingly, induced degradation of MCM10 leads in
parallel to degradation of DNA polymerase-a-primase
with similar decay kinetics. This result is striking, as
DNA polymerase-a-primase is normally a protein complex with a long half-life. Targeting of DNA polymerasea-primase to chromatin by MCM10 has been also
Current Opinion in Cell Biology 2006, 18:130–136
134 Cell regulation
observed in S. pombe [43]. Thus, MCM10 may have a dual
function, first to stabilize DNA polymerase-a-primase
and second to target it to chromatin [42]. Formation of
MCM10 homocomplexes [40] suggests the interesting
possibility that the earlier-assembled MCM10 may serve
as a docking platform for the MCM10–DNA polymerasea-primase complex [42,44]. After replication initiation,
in budding yeast MCM10 seems to travel along with the
replication fork, before being disassembled from chromatin in G2 [42]. In fission yeast, MCM10 has been found to
have a DNA primase activity, which can synthesize RNA
primers (K Fien and J Hurwitz, personal communication),
a result which may explain the observations described
above. In human cells, Mcm10 is recruited to the chromosomal domains 30–60 min before they replicate and
then dissociates after DNA replication has been initiated
[45]. Clearly, MCM10 is a protein in the focus of interest,
and we can expect more exciting data about its dual role
in the near future.
PCNA or Ki67, to discriminate pre-cancerous from noncancer cells [47]. It will be interesting to know whether it
will be possible to use other MCM proteins as well as
other replication initiation factors as both diagnostic and
prognostic tools for cancer.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
of special interest
of outstanding interest
1.
Bell SP, Dutta A: DNA replication in eukaryotic cells.
Annu Rev Biochem 2002, 71:333-374.
2.
Mendez J, Stillman B: Perpetuating the double helix: molecular
machines at eukaryotic DNA replication origins. Bioessays
2003, 25:1158-1167.
3.
Tye BK: MCM proteins in DNA replication. Annu Rev Biochem
1999, 68:649-686.
4.
Kearsey SE, Labib K: MCM proteins: evolution, properties,
and role in DNA replication. Biochim Biophys Acta 1998,
1398:113-136.
5.
Chang VK, Fitch MJ, Donato JJ, Christensen TW, Merchant AM,
Tye BK: Mcm1 binds replication origins. J Biol Chem 2003,
278:6093-6100.
Conclusions
MCM2–7 proteins are to date the best candidates for the
primary helicase opening up the DNA at replication
origins, although the recently identified RecQL4 DNA
helicase seems to function at a similar step to MCM2–7,
and therefore it will be important to determine its functional interaction with MCM2–7 proteins. The contribution of the different MCM2–7 subunits to the helicase
activity and their organization on chromatin remains
unclear and constitutes one major problem in understanding the function of the MCM2–7 complex. Vertebrate organisms appear to have increased the repertoire of
MCM2–7 proteins with the appearance of two additional
members, MCM8 and MCM9. Why do vertebrate cells
need more MCM2–7-like proteins? Is the appearance of
these proteins during evolution a consequence of the
increase in genome complexity or a consequence of the
diversification of differentiation programmes, or both?
MCM10 has a pivotal role regulating DNA polymerase-a-primase function on chromatin during S phase.
Unravelling the biochemical function of MCM10 will
be an important step forward in understanding the structure and function of eukaryotic replication forks.
Recent advances show that MCM proteins can be used as
excellent markers for tumour prediction. These proteins
are down-regulated following cell cycle exit and this
situation can be used to distinguish differentiated from
undifferentiated cells [46]. Loss of the differentiation
state and unscheduled re-entry into the cell cycle is
one hallmark of cancer. The detection of MCM2–7
proteins in tissues can be used to distinguish cells that
have regained unscheduled proliferation activity. In the
past two years a number of reports have described the
expression of MCM2–7 proteins in normal versus cancer
tissues [46–49], and it has been shown that these proteins
can be more reliable markers than classical ones, such as
Current Opinion in Cell Biology 2006, 18:130–136
6.
Chang VK, Donato JJ, Chan CS, Tye BK: Mcm1 promotes
replication initiation by binding specific elements at
replication origins. Mol Cell Biol 2004, 24:6514-6524.
Using chromatin immunoprecipitation and footprint analysis in this report
and [5], it is demonstrated that MCM1 binds specifically to ARS
sequences and that origin activation at different telomeric ARSs responds
differently to reduced MCM1 binding in an MCM1 mutant.
7.
Lutzmann M, Maiorano D, Mechali M: Identification of full genes
and proteins of MCM9, a novel, vertebrate-specific member of
the MCM2-8 protein family. Gene 2005, 362:51-56.
8.
Tsuyama T, Tada S, Watanabe S, Seki M, Enomoto T: Licensing
for DNA replication requires a strict sequential assembly of
Cdc6 and Cdt1 onto chromatin in Xenopus egg extracts.
Nucleic Acids Res 2005, 33:765-775.
This paper provides the first evidence that sequential binding of Cdc6 and
then Cdt1 is required to assemble MCM2-7 proteins onto chromatin and
to allow DNA replication in Xenopus egg extracts. If this order of loading is
reversed, both Cdc6 and Cdt1 still bind to chromatin but recruitment of
MCM2-7 proteins is not permitted.
9.
Bowers JL, Randell JC, Chen S, Bell SP: ATP hydrolysis by ORC
catalyzes reiterative MCM2-7 assembly at a defined origin of
replication. Mol Cell 2004, 16:967-978.
In the yeast Saccharomyces cerevisiae, a mutant of ORC4 defective in
ATP hydrolysis is shown to be lethal. The loading of MCM2-7 complexes
in vitro onto a DNA replication origin is reduced fourfold in this mutant.
These results suggest that robust MCM2-7 loading at DNA replication
origins is essential in vivo.
10. Speck C, Chen Z, Li H, Stillman B: ATPase-dependent
cooperative binding of ORC and Cdc6 to origin DNA.
Nat Struct Mol Biol 2005, 12:965-971.
The footprint of mutated version of recombinant ORC and Cdc6 is
analyzed onto a DNA replication origin in Saccharomyces cerevisiae.
ORC and Cdc6 interact only in the presence of DNA and ATP hydrolysis
by ORC and Cdc6 is required to form an extended footprint onto the DNA
similar to that found during formation of preRC; this process involves
MCM2-7 loading. Moreover a three-dimensional structure of the ORC
complex alone and in complex with Cdc6 is proposed.
11. Maiorano D, Rul W, Mechali M: Cell cycle regulation of the
licensing activity of Cdt1 in Xenopus laevis. Exp Cell Res 2004,
295:138-149.
12. Kulartz M, Knippers R: The replicative regulator protein geminin
on chromatin in the HeLa cell cycle. J Biol Chem 2004,
279:41686-41694.
www.sciencedirect.com
MCM proteins and DNA replication Maiorano, Lutzmann and Méchali 135
This paper and [11] provide evidence that geminin and Cdt1 bind to
chromatin together already before initiation of DNA synthesis in Xenopus
and human cells, providing a mechanism to block origin firing after preRC formation. Binding of geminin to chromatin is dependent upon Cdt1,
suggesting that geminin is targeted to DNA replication origins. Chromatin
immunoprecipitation experiments show that geminin binds at the lamin
B2 replication origin in human cells at a site adjacent to ORC.
13. Lee C, Hong B, Choi JM, Kim Y, Watanabe S, Ishimi Y, Enomoto T,
Tada S, Cho Y: Structural basis for inhibition of the replication
licensing factor Cdt1 by geminin. Nature 2004, 430:913-917.
This paper describes the first three-dimensional structure of a recombinant complex made of fragments of mouse Cdt1 and geminin proteins.
One molecule of Cdt1 is bound to a dimer of geminin via the geminin’s
coiled-coil domain, which is also the dimerization domain of geminin
itself. This paper also suggests that this interaction could block Cdt1’s
ability to interact with the MCM2-7 complex by steric hindrance.
14. Saxena S, Yuan P, Dhar SK, Senga T, Takeda D, Robinson H,
Kornbluth S, Swaminathan K, Dutta A: A dimerized coiled-coil
domain and an adjoining part of geminin interact with two
sites on Cdt1 for replication inhibition. Mol Cell 2004,
15:245-258.
15. Ballabeni A, Melixetian M, Zamponi R, Masiero L, Marinoni F,
Helin K: Human geminin promotes pre-RC formation and DNA
replication by stabilizing CDT1 in mitosis. EMBO J 2004,
23:3122-3132.
This important report establishes a cell-cycle-driving function of geminin
besides its known role in blocking DNA licensing in human cells. Geminin
has to form a complex with Cdt1 during mitosis to protect it from being
degraded and to maintain sufficient levels of Cdt1 to license DNA at the
beginning of the following cell cycle.
16. Laskey RA, Madine MA: A rotary pumping model for helicase
function of MCM proteins at a distance from replication forks.
EMBO Rep 2003, 4:26-30.
17. Ishimi Y: A DNA helicase activity is associated with an MCM4,
-6, and -7 protein complex. J Biol Chem 1997, 272:24508-24513.
This paper reports the first evidence that a DNA helicase activity is
associated with a sub-complex made of MCM4, 6 and 7 proteins.
18. McGeoch AT, Trakselis MA, Laskey RA, Bell SD: Organization of
the archaeal MCM complex on DNA and implications for the
helicase mechanism. Nat Struct Mol Biol 2005, 12:756-762.
19. Pacek M, Walter JC: A requirement for MCM7 and Cdc45 in
chromosome unwinding during eukaryotic DNA replication.
EMBO J 2004, 23:3667-3676.
20. Shechter D, Ying CY, Gautier J: DNA unwinding is an
MCM-complex and ATP-hydrolysis dependent process.
J Biol Chem 2004.
21. Nedelcheva MN, Roguev A, Dolapchiev LB, Shevchenko A,
Taskov HB, Stewart AF, Stoynov SS: Uncoupling of unwinding
from DNA synthesis implies regulation of MCM helicase by
Tof1/Mrc1/Csm3 checkpoint complex. J Mol Biol 2005,
347:509-521.
22. Lee J, Gold DA, Shevchenko A, Dunphy WG: Roles of replication
fork-interacting and Chk1-activating domains from claspin in
a DNA replication checkpoint response. Mol Biol Cell 2005,
16:5269-5282.
23. Calzada A, Hodgson B, Kanemaki M, Bueno A, Labib K:
Molecular anatomy and regulation of a stable replisome
at a paused eukaryotic DNA replication fork. Genes Dev 2005,
19:1905-1919.
In this elegant study a replication fork barrier is used to trap pausing
replication forks. The molecular composition of the forks is then studied
by chromatin immunoprecipitation with antibodies for replication proteins. MCM4 and Cdc45 as well as DNA polymerase-e and DNA primase
are found at the sites of the paused forks.
24. Katou Y, Kanoh Y, Bando M, Noguchi H, Tanaka H, Ashikari T,
Sugimoto K, Shirahige K: S-phase checkpoint proteins Tof1 and
Mrc1 form a stable replication-pausing complex. Nature 2003,
424:1078-1083.
25. Tourriere H, Versini G, Cordon-Preciado V, Alabert C,
Pasero P: Mrc1 and Tof1 promote replication fork progression
and recovery independently of Rad53. Mol Cell 2005,
19:699-706.
www.sciencedirect.com
26. Liku ME, Nguyen VQ, Rosales AW, Irie K, Li JJ: CDK
phosphorylation of a novel NLS-NES module distributed
between two subunits of the MCM2-7 complex prevents
chromosomal rereplication. Mol Biol Cell 2005, 16:5026-5039.
27. Maiorano D, Krasinska L, Lutzmann M, Mechali M: Recombinant
cdt1 induces rereplication of G2 nuclei in Xenopus egg
extracts. Curr Biol 2005, 15:146-153.
28. Li A, Blow JJ: Cdt1 downregulation by proteolysis and geminin
inhibition prevents DNA re-replication in Xenopus. EMBO J
2005, 24:395-404.
29. Arias EE, Walter JC: Replication-dependent destruction of Cdt1
limits DNA replication to a single round per cell cycle in
Xenopus egg extracts. Genes Dev 2005, 19:114-126.
30. Yoshida K, Takisawa H, Kubota Y: Intrinsic nuclear import
activity of geminin is essential to prevent re-initiation of DNA
replication in Xenopus eggs. Genes Cells 2005, 10:63-73.
31. Castellano Md M, Boniotti MB, Caro E, Schnittger A, Gutierrez C:
DNA replication licensing affects cell proliferation or
endoreplication in a cell-type-specific manner. Plant Cell 2004,
16:2380-2393.
32. Thomer M, May NR, Aggarwal BD, Kwok G, Calvi BR:
Drosophila double-parked is sufficient to induce re-replication
during development and is regulated by cyclin E/CDK2.
Development 2004, 131:4807-4818.
This paper together with [27–31] describes the regulation of the Cdt1
protein in Drosophila, Xenopus and plants. Cdt1 is shown to be excluded
from the nucleus in Xenopus by degradation after the initiation of DNA
synthesis, similar to what occurs in human cells. Over-expression of Cdt1
or addition of proteasome inhibitors and depletion of geminin results in
MCM2-7 re-loading onto chromatin and extensive re-replication. Overexpression of Cdc6 does not induce re-replication, suggesting that Cdt1
is rate limiting for the initiation of DNA synthesis in Xenopus egg extracts.
However, in plant cells Cdc6 over-expression does induce re-replication,
suggesting that regulatory mechanisms may also depend upon cell types.
33. Gozuacik D, Chami M, Lagorce D, Faivre J, Murakami Y,
Poch O, Biermann E, Knippers R, Brechot C, Paterlini-Brechot P:
Identification and functional characterization of a new
member of the human Mcm protein family: hMcm8.
Nucleic Acids Res 2003, 31:570-579.
34. Maiorano D, Cuvier O, Danis E, Mechali M: MCM8 is an
MCM2-7-related protein that functions as a DNA helicase
during replication elongation and not initiation. Cell 2005,
120:315-328.
35. Sangrithi MN, Bernal JA, Madine M, Philpott A, Lee J, Dunphy WG,
Venkitaraman AR: Initiation of DNA replication requires the
RECQL4 protein mutated in Rothmund-Thomson syndrome.
Cell 2005, 121:887-898.
36. Volkening M, Hoffmann I: Involvement of human MCM8 in
prereplication complex assembly by recruiting hcdc6 to
chromatin. Mol Cell Biol 2005, 25:1560-1568.
37. Oehlmann M, Score AJ, Blow JJ: The role of Cdc6 in ensuring
complete genome licensing and S phase checkpoint
activation. J Cell Biol 2004, 165:181-190.
38. Yoshida K: Identification of a novel cell-cycle-induced MCM
family protein MCM9. Biochem Biophys Res Commun 2005,
331:669-674.
39. Blanton HL, Radford SJ, McMahan S, Kearney HM, Ibrahim JG,
Sekelsky J: REC, Drosophila MCM8, drives formation of
meiotic crossovers. PLoS Genet 2005, 1:e40.
40. Cook CR, Kung G, Peterson FC, Volkman BF, Lei M: A novel zinc
finger is required for Mcm10 homocomplex assembly.
J Biol Chem 2003, 278:36051-36058.
41. Sawyer SL, Cheng IH, Chai W, Tye BK: Mcm10 and Cdc45
cooperate in origin activation in Saccharomyces cerevisiae.
J Mol Biol 2004, 340:195-202.
42. Ricke RM, Bielinsky AK: Mcm10 regulates the stability and
chromatin association of DNA polymerase-a. Mol Cell 2004,
16:173-185.
The authors use a novel histone association assay to show that assembly of
MCM10 to replication origins occurs in two distinct steps, both dependent
Current Opinion in Cell Biology 2006, 18:130–136
136 Cell regulation
on MCM2-7. MCM10 is shown to be first recruited just after Cdc45 and
second, at a later time point, in a complex with DNA polymerase-a-primase.
Interestingly, the authors found that free MCM10 stabilizes DNA polymerase-a-primase through complex formation. It is likely that the MCM10- DNA
polymerase a-primase complex is recruited through complex formation
with the earlier assembled MCM10 on replication origins.
43. Fien K, Cho YS, Lee JK, Raychaudhuri S, Tappin I, Hurwitz J:
Primer utilization by DNA polymerase a-primase is influenced
by its interaction with Mcm10p. J Biol Chem 2004,
279:16144-16153.
44. Yang X, Gregan J, Lindner K, Young H, Kearsey SE: Nuclear
distribution and chromatin association of DNA polymerase
a-primase is affected by TEV protease cleavage of Cdc23
(Mcm10) in fission yeast. BMC Mol Biol 2005, 6:13.
In this study, the function of MCM10 in fission yeast is addressed by
construction of a conditional allele of MCM10 (cdc23) by insertion of a
cleavage site for the TEV protease into the MCM10 protein. The cleavage
removes a 170-amino-acid C-terminal region of MCM10 and results in a
DNA replication block. Characterization of this phenotype shows that the
chromatin binding and nuclear distribution of the primase subunit of DNA
polymerase-a is affected.
45. Izumi M, Yatagai F, Hanaoka F: Localization of human Mcm10
is spatially and temporally regulated during the S phase.
J Biol Chem 2004, 279:32569-32577.
Current Opinion in Cell Biology 2006, 18:130–136
46. Gonzalez MA, Tachibana KE, Laskey RA, Coleman N:
Control of DNA replication and its potential clinical
exploitation. Nat Rev Cancer 2005, 5:135-141.
47. Schrader C, Janssen D, Klapper W, Siebmann JU, Meusers P,
Brittinger G, Kneba M, Tiemann M, Parwaresch R:
Minichromosome maintenance protein 6, a proliferation
marker superior to Ki-67 and independent predictor of survival
in patients with mantle cell lymphoma. Br J Cancer 2005,
93:939-945.
48. Murphy N, Ring M, Heffron CC, King B, Killalea AG, Hughes C,
Martin CM, McGuinness E, Sheils O, O’Leary JJ: p16INK4A,
CDC6, and MCM5: predictive biomarkers in cervical
preinvasive neoplasia and cervical cancer. J Clin Pathol 2005,
58:525-534.
49. Korkolopoulou P, Givalos N, Saetta A, Goudopoulou A,
Gakiopoulou H, Thymara I, Thomas-Tsagli E, Patsouris E:
Minichromosome maintenance proteins 2 and 5 expression in
muscle-invasive urothelial cancer: a multivariate survival
study including proliferation markers and cell cycle
regulators. Hum Pathol 2005, 36:899-907.
50. Furstenthal L, Kaiser BK, Swanson C, Jackson PK: Cyclin E uses
Cdc6 as a chromatin-associated receptor required for DNA
replication. J Cell Biol 2001, 152:1267-1278.
www.sciencedirect.com