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Biochemical Society Transactions (2009) Volume 37, part 4
Regulation of Cdc45 in the cell cycle and
after DNA damage
Ronan Broderick1 and Heinz-Peter Nasheuer
Cell Cycle Control Group, Centre for Chromosome Biology, Department of Biochemistry, National University of Ireland, Galway, Galway, Ireland
Abstract
The Cdc (cell division cycle) 45 protein has a central role in the regulation of the initiation and elongation
stages of eukaryotic chromosomal DNA replication. In addition, it is the main target for a Chk1 (checkpoint
kinase 1)-dependent Cdc25/CDK2 (cyclin-dependent kinase 2)-independent DNA damage checkpoint signal
transduction pathway following low doses of BPDE (benzo[a]pyrene dihydrodiol epoxide) treatment, which
causes DNA damage similar to UV-induced adducts. Cdc45 interacts physically and functionally with the
putative eukaryotic replicative DNA helicase, the MCM (mini-chromosome maintenance) complex, and
forms a helicase active ‘supercomplex’, the CMG [Cdc45–MCM2–7–GINS (go-ichi-ni-san)] complex. These
known protein–protein interactions, as well as unknown interactions and post-translational modifications,
may be important for the regulation of Cdc45 and the initiation of DNA replication following DNA damage.
Future studies will help to elucidate the molecular basis of this newly identified S-phase checkpoint
pathway which has Cdc45 as a target.
Eukaryotic DNA replication
In all organisms, including humans, accurate chromosomal
DNA replication is essential to maintain stability of the
genome and for normal cell division, with replication of
damaged DNA leading to mutations in the genome and to
the development of diseases such as cancer [1]. Therefore
understanding the molecular mechanisms regulating DNA
replication after DNA damage is of great importance to
biomedical researchers, and detailed knowledge of these
mechanisms may lead to the identification of novel molecular
targets for drug therapies in cancer and other diseases.
Eukaryotic DNA replication occurs during S-phase of
the cell cycle and is a highly regulated process involving
various replication proteins [2–9]. DNA replication is
initiated at distinct origins of replication on chromosomal
DNA. To activate an origin, the multisubunit ORC (origin
recognition complex) must bind to chromatin at origins of
DNA replication [2,6,8]. This ORC–DNA complex serves
as a landing platform for the binding of Cdc (cell division
cycle) 6 and Cdt1 (Cdc10-dependent target 1) [2,6,8]. This
multimeric Cdc6–Cdt1–ORC–DNA complex facilitates the
loading of the MCM (mini-chromosome maintenance) 2–7
complex to chromatin, forming the preRC (pre-replicative
complex) [2,6,8]. Activation of the preRC by CDKs (cyclindependent kinases) and DDK (Dbf4-dependent kinase)
allows the formation of an initiation complex consisting
of RPA (replication protein A), Cdc45 and the GINS
Key words: Cdc45, cell cycle, checkpoint, DNA damage, DNA replication, genome stability.
Abbreviations used: ATM, ataxia telangiectasia mutated; ATR, ATM- and Rad3-related; BPDE,
benzo[a]pyrene dihydrodiol epoxide; Cdc, cell division cycle; CDK, cyclin-dependent kinase; Cdt1,
Cdc10-dependent target; Chk, checkpoint kinase; DDK, Dbf4-dependent kinase; GINS, go-ichini-san (five-one-two-three); MCM, mini-chromosome maintenance; CMG, Cdc45–MCM2–7–GINS;
ORC, origin recognition complex; PCNA, proliferating-cell nuclear antigen; Pol, DNA polymerase;
Pol-prim, DNA polymerase α-primase; preRC, pre-replicative complex; RFC, replication factor C;
RPA, replication protein A.
1
To whom correspondence should be addressed (email [email protected]).
C The
C 2009 Biochemical Society
Authors Journal compilation [go-ichi-ni-san (five-one-two-three)] complex, activating
the replicative helicase complex and causing the unwinding
of the DNA double helix. MCM10 protein facilitates the
chromatin-association of Pol-prim (DNA polymerase
α-primase), which coincides with the initiation of DNA
replication [3,4]. In addition, topoisomerase 1 is recruited to
sites ahead of the progressing replication fork, where it nicks
the DNA backbone in order to relieve tortional stress on the
DNA caused by supercoiling during replication [2].
Subsequently, the first RNA primer is synthesized by
the primase activity of Pol-prim and elongated by its DNA
polymerase activity [2,8,9]. This RNA–DNA is recognized
by RFC (replication factor C) which loads the PCNA
(proliferating-cell nuclear antigen), the replicative sliding
clamp, on to DNA [2,8,9]. RFC and PCNA, together with
RPA, mediate a polymerase switch from Pol-prim to Pol
(DNA polymerase) δ or ε, allowing continuous DNA synthesis on the leading strand and discontinuous DNA
synthesis on the lagging strand [2] (Figure 1). On the
lagging strand, the repetitive priming of Pol-prim followed
by RFC- and PCNA-mediated polymerase switch causes
the formation of Okazaki fragments, which are matured
into a continuous new strand by the actions of RNAse H,
Fen1 (flap endonuclease 1), Dna1, Pol δ and DNA ligase 1
[2,8,9].
Cdc45
The Cdc45 protein is involved in the regulation of eukaryotic
chromosomal DNA replication and is required for the
initiation and elongation steps as well as the co-ordination
of DNA replication [5–9]. Cdc45 was originally identified in
yeast screens as an essential gene involved in the regulation
of DNA replication [10]. Additionally, the CDC45 gene was
shown to genetically interact with the replication factors
MCM5/CDC46, MCM7/CDC47 and ORC [10]. These
Biochem. Soc. Trans. (2009) 37, 926–930; doi:10.1042/BST0370926
Genome Instability and Cancer
Figure 1 Cdc45 function at the replication fork during the
Figure 2 Model of the distinct mechanisms of Cdc45 regulation in
elongation stage of eukaryotic DNA replication
Proposed model for localization and function of human Cdc45 during the
response to low and high levels of UV damage
Low levels of UV- or BPDE-induced DNA adducts inhibit Cdc45 loading
elongation stage of DNA replication. Cdc45 associates with replicative
DNA polymerases, GINS and with the MCM2–7 complex, probably acting
as a molecular tether between the replicative polymerases and the
in a Chk1-dependent manner that does not involve changes in Cdc25A
and CDK2. In contrast, high levels of DNA adducts that cause global
replication blocks activate Chk1/Chk2 and target Cdc25A for degradation.
MCM2–7 complex. Adapted from [9], with permission from Blackwell
Publishing Ltd.
The resulting inhibition of CDK2 activity prevents Cdc45 loading at unfired
origins.
interactions implied a role for Cdc45 in the initiation and
elongation stages of DNA replication [10] (Figure 1).
Cdc45 and the initiation of eukaryotic
DNA replication
Cdc45 plays a crucial role in the initiation of DNA replication. Chromatin association of Cdc45 corresponds well with
origin activation and the progression of DNA replication [11]
(Figure 1), and Cdc45 chromatin association is mediated by
the converging actions of CDKs and DDK [12], implying that
Cdc45 is also crucial in the regulation of initiation of DNA
replication. Moreover, it has been demonstrated that Cdc45
chromatin association is mediated in a DDK-dependent manner, with chromatin association of Cdc45 essential for the progression of DNA replication [12]. As this DDK complex only
exists in late G1 - and S-phase of the cell cycle and the assembly
of Cdc45 into protein complexes containing MCM2–7
proteins is DDK-dependent, this regulation of Cdc45 protein
may then finally control the initiation of DNA replication
[12]. Currently, it is anticipated that the association of Cdc45
with the MCM and GINS complexes are the final steps to activate the replicative helicase in eukaryotic cells, a prerequisite
to initiate DNA replication [6,7,9,13]. These studies imply
that Cdc45 may represent a ‘limiting factor’ in the imitation of
DNA replication and a crucial target for signalling pathways
regulating cell-cycle progression and DNA replication.
Cdc45 and the elongation stage of
eukaryotic DNA replication
Co-immunoprecipitation studies in yeast have revealed
numerous physical interactions between Cdc45 and proteins
involved in the elongation stage of DNA replication,
including Sld3 protein [14]. In yeast, Cdc45 was also purified
as part of a complex containing MCM2 protein [15] and was
shown to interact physically with MCM5 [16], MCM7 [17]
and MCM10 [18] proteins.
Protein–protein interaction studies involving the gene
product of the human homologue of CDC45 corroborate
these data, showing conserved physical interactions with
MCM5 and MCM7 [5], as well as interactions with subunits
of Pol δ and ε [5] and of the GINS complex [5]. In addition,
Cdc45 interacted physically with TopBP1 (topoisomerase
IIβ-binding protein 1) [19] and the p70 subunit of Pol α
[20]. In humans, it has also been demonstrated that Cdc45
interacts with MCM3, MCM7, GINS subunits, RPA, and
Pol δ and ε subunits in a cell cycle-dependent manner [21].
The MCM2–7 complex is the proposed candidate for the
eukaryotic helicase, and, for some time, helicase activity
has only been shown to be present in a subcomplex of the
MCM2–7 complex, consisting of MCM4–6–7 [13]. Recent
results, however, show that the yeast MCM2–7 complex
has helicase activity in vitro depending on buffer conditions
and other factors [22–30]. Interestingly, Cdc45 was purified
in Drosophila as part of a ‘supercomplex’ with MCM2–7
and GINS termed the CMG complex, which was shown
to have helicase activity [13], lending more credence to the
idea that Cdc45 is required for the formation and normal
function of the replication fork. In summary, these studies
imply a crucial role for Cdc45 protein in DNA replication in
human cells, where it is proposed to act as a molecular tether
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Biochemical Society Transactions (2009) Volume 37, part 4
between the MCM2–7 helicase complex and the replicative
DNA polymerases facilitating elongation of replication [5].
Cdc45 in DNA damage-dependent
checkpoint response
Genomic DNA can become damaged via exogenous sources
(UV light or ionizing radiation) or endogenous sources (reactive oxygen species and free radicals) [1,8].
In order to maintain genome stability after DNA damage,
eukaryotic cells have established various processes to repair
DNA lesions and posses mechanisms to initiate DNA
damage-dependent signalling pathways and to establish
DNA damage-dependent checkpoints throughout the cell
cycle, which arrest cells in certain cell cycle phases to facilitate
repair of damaged DNA or apoptosis of the cell [1,2,8,31].
These checkpoint pathways are essential to diminish genome
instability of eukaryotic cells and tumour development in
humans [31–35] (Figure 2).
To this end, cells activate signal transduction pathways mediated by sensor, transducer and effector proteins in response
to DNA damage. These pathways control a wide variety of
processes including cell-cycle arrest and DNA repair pathways, modulate transcriptional activity and may lead to programmed cell death via apoptosis in some cell types [34]. One
class of signal transducers are the PI3K (phosphoinositide
3-kinase)-like kinase family, which include the ATM (ataxia
telangiectasia mutated) and ATR (ATM- and Rad3-related)
proteins in mammals and their homologues in yeast [34]. In
addition, these transducers act upstream in the regulation of
two checkpoint kinases Chk1 and Chk2, which act in a subset
of the damage response involved in cell-cycle regulation
[34]. These proteins are involved in cell-cycle checkpoints
which affect the initiation of replicons via intra-S-phase
checkpoint signalling [35] (Figure 2). The ionizing radiationinduced intra-S-phase checkpoint requires signalling from
ATM through Chk1 and Chk2 to Cdc25A to inhibit cyclin
E/CDK2 following double-strand breaks [35] (Figure 2). Another pathway exists which signals through SMC1 (structural
maintenance of chromosomes 1) protein, although this signalling is poorly understood [35]. A different signalling pathway is activated which inhibits replicon initiation after treatment with UV or by the carcinogen BPDE (benzo[a]pyrene
dihydrodiol epoxide), with the latter inducing bulky adducts
on DNA similar to UV-induced lesions [35,36]. These agents
do not directly induce double-strand breaks and do not signal
through ATM [35,36]. Instead, ATR signals through Chk1 at
low doses of UV and BPDE, which selectively inhibit replicon
initiation and have little effect on DNA chain elongation [35].
In addition, under these conditions, no Cdc25A degradation
occurs and cyclin E/CDK2 is not inhibited [35].
Cdc45 is involved in one such DNA damage-dependent
signal transduction pathway [36]. Recent studies demonstrate
that Cdc45 is the main target of a Chk1-mediated DNA
S-phase checkpoint, which operates via a Cdc25A/CDK2independent mechanism [36]. In this case, DNA damage
induced by BPDE, there is a demonstrated reduction in
chromatin-association of Cdc45, accompanied by Chk1
C The
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Authors Journal compilation activation and the inhibition of DNA synthesis [36]. This was
accompanied by the inhibition of the association between
Cdc45 and MCM7 on the chromatin. Moreover, Cdc45
dissociates from a known origin of replication after BPDE
treatment [36]. Chromatin association of Cdc45 was shown
to reduce in a dose- and time-dependent manner following
BPDE treatment, with low doses inducing Chk1 activation,
followed by the recovery of Cdc45 chromatin association
and DNA synthesis post-treatment [36]. Conversely, high
doses of BPDE induced Chk1 and Chk2 activation, where
chromatin association of Cdc45 and hence DNA synthesis
were found not to recover post-treatment [36].
These results indicate that Chk1 activation negatively
regulates the association of Cdc45 and MCM7 at origins of replication via a pathway which operates independently of
Cdc25A/CDK2 following low-dose BPDE-induced DNA
damage, and that a separate pathway is activated following
high-dose-BPDE treatment [36]. This implies that the Cdc45
protein is an important target of a Chk1-mediated S-phase
checkpoint [23]. Thus these findings raise the question of
the nature and the molecular basis of this newly identified
S-phase checkpoint which has Cdc45 as a target.
Outlook
First, the use of a UV-mimetic agent (BPDE) [36] raises the
question of whether the same signalling pathway is activated
after exposing cells to UV. To address this issue, our group
has analysed chromatin association of Cdc45 following low
and high dose of UV treatment by Western blotting in HeLa
S3 cells, confirming the change in chromatin association of
Cdc45 following DNA damage observed previously when
using BPDE (R. Broderick and H.-P. Nasheuer, unpublished
work).
The Cdc45 protein contains a putative PEST (ProGlu/Asp-Ser-Thr) motif, several D boxes and a KEN
(Lys-Glu-Asn) box [37], all of which are motifs which can be
ubiquitylated. As ubiquitylation may not only regulate protein degradation by the proteasome, but also regulate
protein function [38], the question exists whether Cdc45
is regulated by ubiquitylation, what residues are modified,
and do any modifications occur in a DNA-damage- or cell
cycle-dependent manner. Proteomic analyses of Cdc45 are
on the way to address this question and to study Cdc45
modifications in soluble and chromatin-associated fractions
in cells synchronized at the various cell cycle stages, and cells
treated with low and high doses of UV. These functional
studies will then be followed up using directed mutagenesis
and ectopic protein expression.
In addition, it is currently unknown whether protein–
protein interactions of Cdc45 regulate its function either
during the normal cell cycle or after UV damage.
Bauerschmidt et al. [5,21] extensively analysed the
interactions of Cdc45 with proteins known to be involved
in DNA replication throughout the normal cell cycle.
However, to date, very little data exist about the nature
of protein–protein interactions involving Cdc45 following
DNA damage. Also, the use of proteomic analysis to screen
Genome Instability and Cancer
for novel interaction partners both through the normal cell
cycle and post-UV treatment may yield more information
about the role of Cdc45 in these processes. In conjunction
with this, Bauerschmidt et al. [5] examined the subcellular
localization of Cdc45 throughout the cell cycle. They
describe a mainly nucleoplasmic signal for Cdc45 with a
diffuse staining at G1 -phase, punctuate staining at S- and
G2 -phase and a diffuse signal during mitosis, where the
Cdc45 signal appears distinct from DNA as stained by DAPI
(4 ,6-diamidino-2-phenylindole) [5]. However, no analysis of
the subcellular localization of Cdc45 following UV treatment
has yet been described, which merits further investigation.
Indeed, our group has determined significant rearrangements
in the subcellular localization of Cdc45 post-UV damage (R.
Broderick and H.-P. Nasheuer, unpublished work).
In summary, the Cdc45 protein plays a crucial role in both
the initiation and elongation stages of DNA replication, and
hence plays a central role in the maintenance of genome
stability. The molecular bases of these processes have yet to be
fully elucidated, with the possibilities that post-translational
modifications and protein–protein interactions may regulate
the function of Cdc45 both during the normal cell cycle
and following DNA damage. Further work needs to be
carried out to yield answers to these questions. Such studies
will shed new light on the processes of eukaryotic DNA
replication and the maintenance of genome stability.
Funding
This work was supported by the Irish Council for Research for
Science, Engineering and Technology, the International Association
for the promotion of co-operation with scientists from the New
Independent States of the former Soviet Union, Health Research
Board Ireland and Science Foundation Ireland grants to H.-P.N.
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Received 31 March 2009
doi:10.1042/BST0370926