doi:10.1093/jmcb/mjq052
Journal of Molecular Cell Biology (2011), 3, 13 –22
| 13
Review
Prevention of DNA re-replication in eukaryotic cells
Lan N. Truong and Xiaohua Wu*
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA
* Correspondence to: Xiaohua Wu, E-mail: [email protected]
DNA replication is a highly regulated process involving a number of licensing and replication factors that function in a carefully
orchestrated manner to faithfully replicate DNA during every cell cycle. Loss of proper licensing control leads to deregulated
DNA replication including DNA re-replication, which can cause genome instability and tumorigenesis. Eukaryotic organisms have
established several conserved mechanisms to prevent DNA re-replication and to counteract its potentially harmful effects. These
mechanisms include tightly controlled regulation of licensing factors and activation of cell cycle and DNA damage checkpoints.
Deregulated licensing control and its associated compromised checkpoints have both been observed in tumor cells, indicating
that proper functioning of these pathways is essential for maintaining genome stability. In this review, we discuss the regulatory
mechanisms of licensing control, the deleterious consequences when both licensing and checkpoints are compromised, and
present possible mechanisms to prevent re-replication in order to maintain genome stability.
Keywords: DNA re-replication, cell cycle checkpoints, DNA damage response, Cdt1, DSB repair, genome stability, tumorigenesis
Introduction
To ensure genome stability, DNA must be replicated once and
only once during each cell cycle. Additional rounds of replication
of genomic DNA, or even sections of it, within a given cell cycle
would result in gene amplification, polyploidy and other kinds
of genome instability, which is a hallmark of tumorigenesis
(Schimke et al., 1986; Albertson, 2006; Hook et al., 2007; Cook,
2009). The complex control of DNA replication initiation in eukaryotic organisms is highly conserved in regards to the DNA licensing
factors, which include the origin of replication complex (ORC) proteins, Cdc10-dependent transcript factor 1 (Cdt1), and cell division cycle 6 (Cdc6), as well as their conserved activities to help
initiate DNA replication via the minichromosome maintenance
(MCM) complex (Blow and Dutta, 2005; Arias and Walter, 2007;
Kim and Kipreos, 2007b). It is imperative for cells to tightly regulate Cdt1 and Cdc6 activities to prevent re-initiation of DNA replication in S and G2 phases so that DNA is replicated only once
during a given cell cycle (Blow and Dutta, 2005; Fujita, 2006;
Arias and Walter, 2007; Hook et al., 2007). To this end, multiple
distinct and redundant pathways function to control Cdt1
expression and activity, in particular mechanisms of proteolytic
degradation of Cdt1 as a means to prevent Cdt1-induced
re-replication (Blow and Dutta, 2005; Fujita, 2006; Arias and
Walter, 2007).
It has been described that loss of the licensing control can
induce re-replication and activate the cell cycle checkpoints
(Hook et al., 2007; Cook, 2009). Re-replication leads to the
# The Author (2011). Published by Oxford University Press on behalf of Journal of
Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
accumulation of single-strand DNA (ssDNA) and the formation
of DNA double-strand breaks (DSBs) (Davidson et al., 2006; Liu
et al., 2007), and these DNA lesions activate the ataxia telangiectasia and Rad3 related (ATR) and ataxia telangiectasia mutated
(ATM) checkpoint pathways (Cook, 2009). Cells with intact checkpoints can prevent further re-replication and arrest cell cycle or
induce cell death, thus suppressing the harmful effects caused
by re-replication (Hook et al., 2007; Liu et al., 2007).
Compromising both licensing control and checkpoint pathways
has been observed in various kinds of tumors, suggesting that
DNA re-replication promotes genome instability and tumorigenesis, especially in the absence of functional checkpoint control.
The control of DNA replication to
ensure one round of DNA replication
per cell cycle
The dynamic control of DNA replication initiation is mediated by a
two-step mechanism: (i) formation of the pre-replicative complex
(pre-RC) comprised of ORC1– 6 complex, Cdc6, Cdt1 and MCM2– 7
complex in late mitosis and early G1, and (ii) activation of MCM2–
7 complex to initiate origin firing and DNA replication during S
phase (Bell and Dutta, 2002; Arias and Walter, 2007; Drury and
Diffley, 2009). Formation of the pre-RC complex occurs as a
sequential assembly of the licensing factors Cdt1 and Cdc6 onto
ORC-bound chromatin and the recruitment of the MCM2– 7
complex (Figure 1; Cocker et al., 1996; Donovan et al., 1997;
Maiorano et al., 2000; Nishitani et al., 2000; Rialland et al.,
2002; Randell et al., 2006; Chen et al., 2007). Direct association
of Cdt1 with several components of the MCM2–7 complex involves
14 | Journal of Molecular Cell Biology
Truong and Wu
Figure 1 Regulation of DNA replication during a cell cycle. At the end of the mitosis and G1, Geminin is degraded, and Cdt1 and Cdc6 recruit
MCM2 – 7 to ORC-bound origins to establish the pre-RC complex. At the onset of S-phase, CDKs and Cdc7/Dbf4 kinase (DDK) establish the pre-IC
complex by recruiting Cdc45 and the GINS complex to MCM2 – 7. With Cdc45 and GINS as accessory factors, MCM2– 7 unwinds DNA, followed by
recruitment of replication machinery to start DNA replication. As MCM2 – 7 moves away from origins, pre-RCs are disassembled. In S and G2,
Geminin is expressed to inhibit Cdt1 activity and Cdt1 is also subject to proteasomal degradation, while Cdc6 is exported to cytoplasm. These
mechanisms prevent reassembly of pre-RC complex on already fired origins and subsequent re-replication within one cell cycle.
both the N-terminus and C-terminus of Cdt1, where the very
extreme C-terminus contains a conserved MCM complex binding
motif that binds MCM6 (Yanagi et al., 2002; Ferenbach et al.,
2005; Teer and Dutta, 2008; Khayrutdinov et al., 2009; Jee et al.,
2010; Wei et al., 2010). Additional studies to further clarify the
mechanism for Cdt1-mediated recruitment of Mcm2–7 to chromatin revealed the requirement for MCM9, which directly binds to
Cdt1 and forms a stable complex to promote the interaction
between Cdt1 and MCM2–7 complex. Thus, MCM9 is suggested
to function as a ‘colicenser’ of DNA replication with Cdt1
(Lutzmann and Mechali, 2008). In vitro analysis suggested that
the association of Cdt1 and Cdc6 with origins is reduced when
MCMs are loaded onto origins (Tsakraklides and Bell, 2010).
Although the MCM2–7 complex is recruited to form the pre-RC
complex in G1, activation of MCM proteins by the
Cyclin-Dependent Kinase (CDK), Cdk2, and the Dbf4-Dependent
Kinase (DDK), Cdc7, to establish the pre-initiation complex
(pre-IC) does not actually occur until the onset of S phase (Bell
and Dutta, 2002; Arias and Walter, 2007; Drury and Diffley,
2009). At the G1/S transition, Cdk2 and Cdc7-mediated phosphorylation events, along with the activities from other players
such as MCM10, are required for recruiting Cdc45 and GINS onto
MCM2–7, which activate MCM2–7 and promote its DNA helicase
activities (Wohlschlegel et al., 2002; Pacek and Walter, 2004;
Gambus et al., 2006; Moyer et al., 2006). Subsequent recruitment
of RPA, DNA polymerase a, RFC, PCNA, and DNA polymerase d
initiates DNA replication (Waga and Stillman, 1998). In addition
to its role to recruit MCM2–7 to chromatin for establishing
pre-RCs, recent studies demonstrate that Cdt1 also participates
in the activation of MCM2–7 complex by directly associating with
Dbf4 of the Cdc7–Dbf4 complex contributing to the recruitment
of Cdc45 to MCM2–7 (Ballabeni et al., 2009). Furthermore, Cdt1
was shown to stimulate MCM2–7 helicase activity through in
vitro gel shift assays combined with DNA helicase assay,
suggesting a role for Cdt1 to promote efficient DNA unwinding
during replication (You and Masai, 2008). Once DNA replication
is initiated, the pre-RCs are disassembled as MCM2–7 moves
away from the origins, releasing Cdt1 and Cdc6 from the origins,
which are subject to proteolytic degradation and nuclear export
[(Aparicio et al., 1997; Blow and Dutta, 2005; Arias and Walter,
2007), Figure 1]. These mechanisms are thought to help prevent
loading of de novo MCM2–7 complex onto the already fired
origins to reassemble pre-RCs leading to DNA re-replication
(Blow and Dutta, 2005; Arias and Walter, 2007; Xouri et al., 2007).
The regulation of replication
licensing factors during the cell
cycle
In order to maintain proper licensing control, various pathways
are utilized to tightly regulate the expression and activity of the
licensing factors during the cell cycle so that unscheduled DNA
licensing is prevented in S and G2 after DNA replication is initiated
(Blow and Dutta, 2005; Arias and Walter, 2007; Cook, 2009). In
Saccharomyces cerevisiae, Cdc6 is degraded, and both Cdc6
and Orc6 are inhibited by binding to Clb proteins (Drury et al.,
1997; Mimura et al., 2004; Wilmes et al., 2004), while Cdt1 is primarily inhibited by nuclear export with MCM2– 7 in a
CDK-dependent manner (Nguyen et al., 2000, 2001; Arias and
Walter, 2007). In contrast, Schizosaccharomyces pombe utilizes
proteolytic degradation to regulate both Cdc6 and Cdt1 in S
and G2, in which Cdt1 is degraded through the Cul4– Ddb1–
Cdt2 E3 ubiquitin ligase complex (Jallepalli et al., 1997;
Gopalakrishnan et al., 2001; Hu and Xiong, 2006; Ralph et al.,
2006). Proteolytic degradation of Cdt1 is a conserved regulatory
mechanism that extends to higher eukaryotes including
Caenorhabditis elegans, Drosophila, Xenopus, and mammals
(Arias and Walter, 2007; Cook, 2009).
Higher eukaryotes also negatively regulate Cdt1 via Geminin,
which binds to and sequesters Cdt1 on chromatin during S and
G2, thus inhibiting Cdt1 association with MCM2– 7 and preventing
pre-RC reassembly within one cell cycle (Wohlschlegel et al.,
2000; Tada et al., 2001; Lee et al., 2004; Maiorano et al.,
Prevention of DNA re-replication in eukaryotic cells
2004). The significance of Geminin-mediated Cdt1 inhibition is
evident by data demonstrating that loss of Geminin alone is sufficient to induce DNA re-replication (Mihaylov et al., 2002; Zhu
et al., 2004; Melixetian et al., 2004; Li and Blow, 2005; Kerns
et al., 2007). Interestingly, additional studies also suggest a positive role of Geminin to promote Cdt1-mediated Mcm2– 7 chromatin loading in G1. Although exact mechanisms which regulate
these ‘licensing permissive’ Cdt1– Geminin activities have yet to
be fully elucidated, structure analysis of the Cdt1– Geminin interaction reveal a dynamic, complex regulation in which the stoichiometry of the Cdt1–Geminin complex determines its activity to
recruit Mcm2–7 for licensing or to inhibit pre-RC formation
(Lutzmann et al., 2006; De Marco et al., 2009). It is proposed that
elevated Geminin levels in S-phase convert the Cdt1–Geminin
complex to a licensing-defective state (De Marco et al., 2009).
While Cdt1 is negatively regulated by proteolysis and Geminin
binding, positive regulation of Cdt1 occurs through E2F-mediated
transcriptional activation during the cell cycle (Karakaidos et al.,
2004; Yoshida and Inoue, 2004) and through acetylation by
HDAC11 and HBO1 (human acetylase binding to Orc1) (Iizuka
et al., 2006; Miotto and Struhl, 2008; Glozak and Seto, 2009).
Acetylation of Cdt1 positively modulates its activity to facilitate
licensing (Miotto and Struhl, 2010) as well as prevents Cdt1
from ubiquitination and subsequent proteasomal degradation
(Glozak and Seto, 2009).
Cdt1 degradation during the cell
cycle
The evolution of multiple distinct and redundant pathways for
Cdt1 proteolysis suggests the importance for inactivating Cdt1
function during S phase and G2 to prevent re-replication (Fujita,
2006; Arias and Walter, 2007; Hook et al., 2007). One degradation pathway is through the SCF – Skp2 E3 ubiquitin ligase
complex, in which Cdt1 proteolysis is regulated by Cdk activity.
In humans, Cdk2 and Cdk4 have both been shown to interact
with Cdt1 on its N-terminus Cy motif (residues 67 – 69) and phosphorylate Cdt1 at residue Thr-29, thus recruiting the SCF– Skp2
complex to Cdt1 for inducing Cdt1 degradation during S and G2
(Figure 2; Li et al., 2003, 2004; Kondo et al., 2004; Sugimoto
et al., 2004; Nishitani et al., 2006). Cdt1 mutants defective for
Skp2 interaction or Cdk-mediated phosphorylation were found
to still undergo degradation in S phase (Takeda et al., 2005;
Nishitani et al., 2006), leading to the discovery of an additional
pathway for Cdt1 proteolysis utilizing the Cul4 – Ddb1– Cdt2 E3
ubiquitin ligase complex (Hu et al., 2004; Takeda et al., 2005;
Arias and Walter, 2006; Jin et al., 2006; Nishitani et al., 2006;
Senga et al., 2006; Kim and Kipreos, 2007a).
Cdt1 degradation through the Cul4 – Ddb1– Cdt2 E3 ubiquitin
ligase complex is mediated by proliferating cell nuclear antigen
(PCNA) (Figure 2; Arias and Walter, 2006; Higa et al., 2006;
Hu and Xiong, 2006; Nishitani et al., 2006; Senga et al., 2006).
In the study using Xenopus extracts, direct binding of PCNA to
Cdt1 only occurs on chromatin when PCNA is loaded onto chromatin during DNA replication, and thus Cdt1 degradation is coupled
with DNA replication (Arias and Walter, 2006; Havens and Walter,
Journal of Molecular Cell Biology
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Figure 2 Degradation of Cdt1 during the cell cycle and after DNA
damage. In S phase, Cdt1 degradation occurs by the SCF –Skp2 and
by the PCNA – Cul4– Ddb1 – Cdt2 pathways. Cyclin A/Cdk2 associates
with Cdt1 on its Cy motif and phosphorylates Cdt1 at residue
Thr-29, followed by recruitment of the SCF– Skp2 E3 ubiquitin
ligase complex. PCNA binds to Cdt1 on its PIP motif, leading to recruitment of Cul4– Ddb1 – Cdt2 E3 ubiquitin ligase complex. Cdt2 recognizes the degron motif (D) adjacent to the PIP box on Cdt1, which
allows for binding of Cdt1 with Cdt2 thus promoting E3 ligase activity
of the Cul4– Ddb1– Cdt2 complex. Following DNA damage by UV/IR
irradiation, PCNA – Cul4 –Ddb1 – Cdt2 mediates Cdt1 degradation on
chromatin in a similar manner as S-phase-induced degradation.
2009). The association of Cdt1 with PCNA involves the PCNAinteracting protein (PIP) motif of the N-terminus of Cdt1, and
mutations within this highly conserved region stabilize Cdt1
protein levels and induce re-replication (Arias and Walter, 2006;
Hu and Xiong, 2006; Nishitani et al., 2006; Senga et al., 2006).
The interaction of PCNA with Cdt1 recruits the Cul4–Ddb1–Cdt2
complex to Cdt1, which contains a ‘degron’ motif located four residues downstream of the Cdt1 PIP motif that is recognized by Cdt2
and targets Cdt1 for degradation (Havens and Walter, 2009).
Depletion of any one of the Cul4–Ddb1–Cdt2 complex members
leads to Cdt1 stabilization and re-replication (Jin et al., 2006;
Lovejoy et al., 2006; Hall et al., 2008). It is proposed that
PCNA-mediated degradation of Cdt1 through Cul4–Ddb1–Cdt2
provides a means for rapid degradation of chromatin-bound Cdt1
during S to prevent re-replication (Arias and Walter, 2006, 2007).
While both SCF–Skp2 and PCNA–Cul4–Ddb1–Cdt2 mediate
degradation of Cdt1 in S phase redundantly, Nishitani et al. elegantly demonstrated the distinct functions of these pathways
during the cell cycle. Utilizing Cdt1 mutants specifically defective
for SCF–Skp2 binding (Cdt1 Cy motif mutant) and for Cul4–
Ddb1–Cdt2 association (Cdt1 PIP motif mutant) in cells synchronized for early S, mid-S, and G2, they showed that Cul4–Ddb1–
Cdt2 functions to degrade Cdt1 only in S phase, whereas
SCF–Skp2 acts in both S and G2 (Nishitani et al., 2006). Thus,
SCF–Skp2 and PCNA–Cul4–Ddb1–Cdt2 function independently of
one another to degrade Cdt1, and SCF–Skp2 is specifically mediated
by Cdk-dependent cell cycle regulation (Hook et al., 2007).
Additional means to negatively regulate licensing factors
include Cul4-mediated nuclear export of Cdc6 through CKI-1
(Kim et al., 2007), caspase-3-mediated cleavage of Cdc6
(Pelizon et al., 2002), and APC/CCdh1-mediated proteolysis of
Cdt1 (Sugimoto et al., 2008). While APC/CCdh1-mediated
16 | Journal of Molecular Cell Biology
ubiquitination and degradation of Cdt1 has been demonstrated to
occur in both in vivo and in vitro systems (Sugimoto et al., 2008),
the exact biological contributions of this pathway has yet to be
fully elucidated with respect to the SCF – Skp2 and PCNA –Cul4 –
Ddb1– Cdt2 pathways.
The regulation of Cdt1 following
DNA damage
In addition to degradation during the cell cycle, Cdt1 proteolysis
in response to DNA damage has also been demonstrated in a
number of organisms (Higa et al., 2003; Ralph et al., 2006; Hall
et al., 2008; Cook, 2009). In mammalian cells, Cdt1 degradation
upon DNA damage caused by both UV and IR has been shown
to be predominantly mediated by the PCNA – Cul4 – Ddb1– Cdt2
pathway, although some evidence also suggests involvement of
the SCF– Skp2 pathway in the UV-induced Cdt1 degradation
(Higa et al., 2003; Hu et al., 2004; Kondo et al., 2004; Ralph
et al., 2006; Hall et al., 2008). Depletion of PCNA, PCNA inhibition
by p21, and the Cdt1 mutants defective for PCNA binding all lead
to stabilized Cdt1 levels under conditions of UV damage (Arias
and Walter, 2006; Hu and Xiong, 2006; Senga et al., 2006). As
well, depleting Cul4, Ddb1, or Cdt2 suppresses Cdt1 degradation
from UV and IR (Higa et al., 2003, 2006; Hu et al., 2004; Jin et al.,
2006). As shown in Figure 2, it was proposed that PCNA – Cul4 –
Ddb1– Cdt2-mediated, damage-induced degradation of Cdt1
occurs when Cdt1 associates with PCNA on chromatin, thus providing a means for rapid destruction of Cdt1 on DNA in response
to DNA damage (Arias and Walter, 2007; Hook et al., 2007).
Further analysis will be needed to address the mechanisms of
how damage-induced Cdt1 degradation occurs outside of
S-phase. It was described that IR induces Cdt1 degradation in
G1, and this degradation also requires Cul4 activity and PCNA
(Higa et al., 2003, 2006). Since PCNA chromatin loading is
usually associated with replication forks, more detailed analysis
will be needed to address how Cdt1 is degraded through the
PCNA – Cul4– Ddb1– Cdt2 pathway when IR is induced in G1. It is
generally believed that PCNA – Cul4– Ddb1– Cdt2-mediated Cdt1
degradation is independent of checkpoints (Arias and Walter,
2007). IR-induced Cdt1 proteolysis in mammalian cells does not
depend on the ATM-Chk2 and ATR-Chk1 pathways, but Kondo
et al. described that UV-induced Cdt1 degradation is caffeinesensitive, suggesting that ATR/ATM may be involved (Higa
et al., 2003; Kondo et al., 2004). In fission yeast, damage-induced
Cdt1 turnover is also dependent on Ddb1 and Cdt2, but independent of checkpoint genes Rad3 and Cds1 (ATR and Chk2 homologues) (Ralph et al., 2006). It still remains possible that
multiple pathways, which can be checkpoint-dependent or
-independent and act in a cell-cycle-regulated manner, are
involved in damage-induced Cdt1 degradation.
Damage-induced Cdt1 degradation may also contribute to the
prevention of DNA re-replication. Upon DNA damage, checkpoints
are activated and consequently Cdk activities are reduced so
that cell cycle is arrested, meanwhile reduced Cdk activity in G2
may allow reassembly of pre-RCs on already fired origins
(Hayles et al., 1994; Dahmann et al., 1995; Itzhaki et al., 1997).
Truong and Wu
Damage-induced Cdt1 degradation is thus important for the prevention of pre-RC reassembly on already fired origins. In addition,
damage-induced Cdt1 degradation may also be involved in the
suppression of replication initiation in response to DNA
damage, as Cdt1 is important for promoting the activation of
MCM2– 7 at the onset of DNA replication in addition to its licensing roles (You and Masai, 2008; Ballabeni et al., 2009).
Consistently, it was described that IR-induced reduction of Cdt1
levels reversely correlates with S-phase recovery (Higa et al.,
2003). Taken together, multiple mechanisms of Cdt1 regulation
by proteasomal degradation during the cell cycle and following
DNA damage all function to ensure proper licensing control and
proper DNA replication.
Cdt1 overexpression leads to DNA
re-replication
DNA re-replication can be induced when the regulation of licensing control factors such as Cdt1 and Cdc6, or the control of
Cdk activities is impaired during the cell cycle (Blow and
Dutta, 2005; Machida et al., 2005; Arias and Walter, 2007).
Cdt1 overexpression alone can induce re-replication in higher
eukaryotes (Vaziri et al., 2003; Maiorano et al., 2005).
Likewise, depletion of the Cdt1-binding inhibitor Geminin is
also sufficient for re-replication (Tada et al., 2001; Mihaylov
et al., 2002; Melixetian et al., 2004; Li and Blow, 2005). One
mechanism by which Cdt1 overexpression can induce
re-replication is thought to be through its activity to recruit
MCM2– 7 onto chromatin and promote pre-RC reassembly
(Cook et al., 2004). Geminin inhibits Cdt1 by preventing
its interaction with MCM2– 7 (Wohlschlegel et al., 2000),
and thus inactivation of Geminin allows non-degraded Cdt1
species in S and G2 to reassemble pre-RCs causing
re-replication. However, one study showed that the Cdt1
mutants deleted for the C-terminal MCM2– 7 binding sites are
still capable to induce re-replication (Teer and Dutta, 2008).
Although it is proposed that the binding of these Cdt1
mutants with PCNA and Cdks through the N-terminus of Cdt1
may titrate away PCNA and Cdks to interact with endogenous
Cdt1 causing re-replication (Teer and Dutta, 2008), additional
complexity and intricacies may be involved by which Cdt1 overexpression leads to re-replication.
DNA re-replication induces the
activation of checkpoint pathways
Since DNA re-replication would inevitably lead to genome
instability, mechanisms to suppress re-replication as a means
to prevent potentially harmful genome instabilities caused by
re-replication would be beneficial to the cell. It has been
addressed by a number of laboratories that cell-cycle checkpoint
is activated when re-replication is induced. In S. cerevisiae, combining mutations in ORC, Cdc6, and MCM2 – 7 causes DNA
re-replication and activates checkpoints (Archambault et al.,
2005). In the Xenopus cell free system, a caffeine-sensitive phosphorylation of Chk1 occurs when re-replication is induced (Li and
Prevention of DNA re-replication in eukaryotic cells
Blow, 2005). In mammalian cells, it was initially observed by Dr.
Dutta’s laboratory that overexpression of Cdt1 induces
activation of the ATM/ATR-dependent checkpoint (Vaziri et al.,
2003).
One important biological function of re-replication-induced
checkpoint activation is to arrest cell cycle or to eliminate cells
with over-replicated DNA by apoptosis or senescence (Figure 3).
Re-replicating yeast cells show a discrete cell cycle arrest
(Archambault et al., 2005). Similarly, in Xenopus egg extracts,
extensive re-replication leads to head-to-tail collision of
re-replication forks with normal replication forks resulting in
DNA fragmentation and DSB formation, which activates checkpoint pathways and blocks further cell cycle progression
(Davidson et al., 2006). In mammalian cells, H2AX phosphorylation is readily observed when re-replication is induced by Cdt1
overexpression or Geminin inactivation (Melixetian et al., 2004;
Zhu et al., 2004; Zhu and Dutta, 2006; Liu et al., 2007),
suggesting that re-replication also causes DSB accumulation in
mammalian cells. Depending on the cellular background, activation of the ATM and ATR-checkpoint pathways induces G2/M
arrest, senescence or apoptosis upon DNA re-replication [(Vaziri
et al., 2003; Melixetian et al., 2004; Zhu et al., 2004) and
Truong and Wu, unpublished results]. The BRCA1-mediated
Fanconi anemia pathway is also suggested to be involved in the
G2/M checkpoint activation (Zhu and Dutta, 2006).
Journal of Molecular Cell Biology
| 17
ATR-mediated checkpoint pathway
plays an important role in
preventing DNA re-replication when
the licensing control is defective
Intriguingly, when the licensing control is disrupted in mammalian
cells by Cdt1 overexpression, extensive re-replication occurs only
in certain tumor cell lines, whereas in primary cell lines and a
number of tumor cell lines (re-replication non-permissive), significant re-replication is not induced while checkpoints are activated
(Vaziri et al., 2003; Tatsumi et al., 2006; Liu et al., 2007).
ATM and ATR are two related checkpoint kinases involved in
detecting abnormal DNA structures and initiating checkpoint activation (Abraham, 2001; Shiloh, 2001). However, these two
kinases respond to different DNA damage signals. Whereas
ATM is activated by DSBs, ATR is activated by ssDNA accumulation at stalled replication forks (Cimprich and Cortez, 2008).
Significantly, when Cdt1 is overexpressed in primary cell lines
and in those tumor cell lines that are resistant to Cdt1-induced
re-replication, inactivation of ATR and its downstream kinase
Chk1 leads to extensive re-replication, while inactivation of ATM
or Chk2 does not promote such re-replication (Liu et al., 2007).
Impaired expression of Rad17 or ATRIP, required for ATR activation (Cimprich and Cortez, 2008), both showed a similar
effect to permit Cdt1-induced re-replication as seen with ATR
Figure 3 The S-phase checkpoint prevents re-replication caused by disruption of the licensing control. Cdt1 is required for initial assembly of
pre-RC complex, and Cdt1 overexpression leads to reassembly of pre-RC complexes on already fired origins. Uncoupling of MCM unwinding from
DNA synthesis at the initiation of re-replication results in ssDNA accumulation, which in turn activates the S-phase checkpoint (Liu et al., 2004).
DSBs are accumulated when stalled re-replication forks are collapsed or when re-replicating forks collide with other forks. In cells with functional checkpoint regulation, activated S-phase checkpoint inhibits re-replication, so that re-replication is suppressed or prevented. Activated
checkpoints also induce cell cycle arrest, as well as apoptosis and senescence when the amount of re-replication-induced DNA lesions exceeds
cellular repair capacity. Compromised checkpoints, combined with disrupted licensing control, lead to overt re-replication which results in
genome instability and tumorigenesis.
18 | Journal of Molecular Cell Biology
deficiency. These observations suggest that the ATR-mediated
S-phase checkpoint acts as a surveillance barrier to prevent
DNA re-replication when the licensing control is impaired in the
‘re-replication non-permissive’ cells lines. This S-phase
checkpoint-mediated suppression of DNA re-replication confers
another layer of protection on top of the licensing control, ensuring one round of DNA replication per cell cycle (Figure 3).
Further studies showed that when the licensing control is
impaired, ATM is activated but its activation occurs later than
that of ATR (Liu et al., 2007). Soon after Cdt1 overexpression,
RPA-bound ssDNA is accumulated, activating ATR prior to the
detection of extensive re-replication, whereas ATM activation
occurs when DSBs are detected at much later stages. This observation is consistent with the hypothesis that ATR is activated
when the initial steps of DNA re-replication are detected, and
this activation in turns suppresses further DNA re-replication.
Given the critical roles of the S-phase checkpoint in the suppression of DNA re-replication, what are the signals triggering
the activation of the S-phase checkpoint upon loss of replication
licensing? When Cdt1 is overexpressed or Geminin expression is
lost, Cdt1 re-loads MCM proteins onto replication forks and
reassembles pre-RCs in S-phase (Figure 3). Inhibition of
re-replication initiation by overexpressing p27 or by inhibiting
Cdc7 kinase activity prevents S-phase checkpoint activation (Liu
et al., 2007), suggesting that re-replication initiation is required
for S-phase checkpoint activation, while Cdt1-mediated reassembly of pre-RCs is not sufficient. Further analysis demonstrated that
at the onset of re-replication, MCM-mediated DNA unwinding is
uncoupled from DNA synthesis, causing ssDNA accumulation at
re-replication forks [(Liu et al., 2007), Figure 3]. These
RPA-bound ssDNA serve as initial signals to activate the
ATR-mediated S-phase checkpoint, which is supported by the
observation that suppressing MCM helicase activity prevents
ssDNA accumulation and attenuates the checkpoint activation
(Liu et al., 2007). Re-replication-induced uncoupling of DNA
unwinding and DNA synthesis is likely due to the helicase activity
that exceeds the rate or capacity of DNA polymerases to synthesize DNA, as re-replication is not a scheduled event. These
studies suggest a critical mechanism to activate the S-phase
checkpoint at the initiation of re-replication.
ATR-mediated S-phase checkpoint targets different downstream effector proteins to mediate the inhibition of DNA
re-replication. Activated ATR directly acts on DNA replication
machinery to inhibit re-replication through phosphorylating replication factors such as RPA2 and MCM2, or indirectly suppresses
DNA replication by modulating the activities of Rb or p53 (Vaziri
et al., 2003; Lee et al., 2007; Liu et al., 2007). Prevention of
DNA re-replication thus requires the intact ATR pathways including sensors, transducers, and effectors. Therefore, it is reasonable to predict that defects in one or more components of the
ATR-dependent S-phase checkpoint pathway, which often
occurs in tumor cells, would cause loss of re-replication suppression function and allow extensive re-replication upon loss of replication licensing control (Figure 3). This provides explanations as
to why overexpression of Cdt1 or inactivation of Geminin causes
extensive re-replication in certain tumor cell lines, but not in
others and in primary cell lines.
Truong and Wu
Inhibition of DNA re-replication by the S-phase checkpoint
pathway is conserved in various organisms. In S. cerevisiae,
deficiency of Mec1 (the ATR homologue) and Rad17 leads to significantly more extensive re-replication when the licensing control
is impaired (Archambault et al., 2005). Similarly, Chk1 activation
antagonizes Cdt1-induced re-replication in Xenopus nuclear
extracts (Li and Blow, 2005).
It was described that Cdt1 and Cdc6 are destabilized when
re-replication is induced by Geminin inactivation or Cdt1 overexpression (Hall et al., 2008). This feedback regulation minimizes the extent of re-replication by proteolysis of the
licensing factors, thereby protecting genome stability. It was
demonstrated that this re-replication-induced Cdt1 degradation
requires the PCNA-binding site of Cdt1 and Cul4– Ddb1– Cdt2
ubiquitin ligase, suggesting that it uses the same pathway
for S-phase- and damage-induced Cdt1 ubiquitination and
degradation (Hall et al., 2008). Presumably, this PCNA – Cul4 –
Ddb1– Cdt2 degradation pathway is independent from the
ATR/ATM checkpoint pathways (Higa et al., 2003; Arias and
Walter, 2005). We thus propose that accumulated ssDNA
upon re-replication may recruit PCNA chromatin binding, triggering the activation of Cul4 – Ddb1– Cdt2 pathway to degrade
Cdt1. Re-replication-induced degradation of Cdc6 is less clear,
but it is known that this degradation requires the Huwe1 ubiquitin ligase (Hall et al., 2008). These observations suggest
that in addition to the re-replication suppression function
mediated
by
the
ATR-checkpoint
pathway,
an
ATR-independent mechanism involving direct degradation of
the licensing factors may also have a role in limiting the
extent of re-replication.
DNA re-replication and DSB repair
DSBs are accumulated when re-replication is induced (Figure 3).
This can be caused by collapse of stalled re-replication forks or
collision of new re-replication forks with existing replication/
re-replication forks (Figure 3; Davidson et al., 2006; Liu et al.,
2007). ATM is activated when DSBs are detected upon loss of
the licensing control. In addition to its role in inducing cell cycle
arrest, apoptosis and senescence, ATM may also be involved in
promoting DSB repair when re-replication is induced.
Inactivation of the Mre11 complex results in accumulation of
more DSBs, suggesting that this complex likely participates in
the repair of DSBs caused by DNA re-replication (Lee et al.,
2007). Yeast mutants impaired in the licensing control and
Rad52 function are synthetic lethal (Archambault et al., 2005),
implying that HR-mediated DSB repair may be involved in repairing re-replication-associated DSBs to maintain cell viability.
Despite these observations, the exact mechanism of how DSBs
are repaired during re-replication is still not clear.
Suppression of re-replication plays critical roles in preventing
genome instability when mutations and defects are present in
the replication control pathways. However, such mechanisms
may be more important for a normal cell to cope with mistakes
at replication onset and/or during replication. For instance, reassembly of preRCs may occur at one or more origins at fault in a
normal cell. In response to such mistakes, inhibition of
Prevention of DNA re-replication in eukaryotic cells
re-replication immediately after DNA unwinding would limit
re-replication to a minimal extent, possibly right after the synthesis of RNA/DNA primers or small stretches of DNA at origins.
Under this circumstance, checkpoint-activated repair pathways
would be able to remove these limited duplicated sequences
and repair re-replication-associated lesions, so that DNA lesions
caused by transient loss of DNA replication control are fixed
and a normal cell cycle can be restored. Checkpoint-induced
cell cycle arrest, apoptosis, or senescence would be only
induced when re-replication-associated DNA lesions exceed the
repair capacity of a normal cell.
DNA re-replication and
tumorigenesis
Deregulated overexpression of Cdt1 and Cdc6 was observed in
various tumor samples. In a set of analysis, 75 cases of non-small
cell lung carcinomas and adjacent healthy lung tissue were examined for the expression levels of Cdt1 and Cdc6 (Karakaidos et al.,
2004). Strikingly, overexpression of Cdt1 and Cdc6 (more than
4-fold) was observed in 43 and 50% of neoplasms, respectively.
Co-overexpression of Cdt1, Cdc6, and E2F1 is common. It has
been shown that E2F1 and E2F2 are important transcription activators of Cdt1 and Cdc6 (Hateboer et al., 1998; Yan et al., 1998;
Karakaidos et al., 2004; Yoshida and Inoue, 2004), and overexpression of E2F family members is thus likely a contributing mechanism for Cdt1 and Cdc6 overexpression in tumors. Other studies
revealed that gene amplification is another source leading to Cdt1
and Cdc6 overexpression (Liontos et al., 2007). Overexpression of
Cdt1 and/or Cdc6 has also been documented in mantle cell lymphoma, colon cancer and head-and-neck carcinomas (Karakaidos
et al., 2004; Pinyol et al., 2006; Liontos et al., 2007). In mouse
models, Cdt1 overexpression predisposes for malignant transformation (Seo et al., 2005). These studies suggest that deregulated overexpression of Cdt1 and Cdc6 and its associated
re-replication are closely linked to tumorigenesis.
To investigate whether deregulated overexpression of Cdt1 and
Cdc6 is an active driving force for tumorigenesis rather than a
mere reflection of increased proliferation rate in tumors, the
expression levels of Cdt1 and Cdc6 was examined at different cancerous and precancerous stages of tumorigenesis of lung, colon,
and head-and-neck cancer (Liontos et al., 2007). Overexpression
of Cdt1 and Cdc6 was detected at mRNA levels by 2-fold in the
hyperplasia when compared with the adjacent normal tissues,
while protein levels of Cdt1 and Cdc6 are elevated at least
4-fold in dysplasia and carcinomas. No correlation of elevated
expression of the proliferation marker Ki-67 with that of Cdt1
and/or Cdc6 was detected (Liontos et al., 2007). It is thus hypothesized that overexpression of Cdt1 and/or Cdc6 promotes DNA
re-replication, leading to genome instability and DNA damage
responses. The activated checkpoints serves as an anti-tumor
barrier, which induces senescence and apoptosis to eliminate
cells with severe DNA lesions associated with DNA re-replication.
Loss of p53 and/or other genome caretaker genes abrogated the
antitumor barriers and resulted in tumorigenesis. Consistently,
loss of p53 was frequently observed in the tumors with
Journal of Molecular Cell Biology
| 19
overexpressed Cdt1 and Cdc6 (Karakaidos et al., 2004; Pinyol
et al., 2006). In the mouse transgenic model, overexpression of
Cdt1 in thymocytes exhibited normal T-cell development, but
developed thymic lymphoblastic lymphoma when crossed to
p53 null mice (Seo et al., 2005).
Activation of checkpoints at the early stages of tumorigenesis
is not only limited to the cancer development that is associated
with overexpression of Cdt1 and Cdc6. It has been described
that activation of DNA damage checkpoint response is a general
consequence of oncogene activation (Gorgoulis et al., 2005;
Venkitaraman, 2005; Bartek et al., 2007). Various oncogenes,
such as H-RasV12, cyclin E, and Cdc25A, induce unscheduled
DNA replication or DNA re-replication at pre-cancerous stages of
tumorigenesis, generating signals to activate DNA damage checkpoint response, and subsequent loss of checkpoint barriers leads
to tumorigenesis (Bartkova et al., 2005, 2006; Di Micco et al.,
2006). These studies suggest that loss of replication control is a
common phenomenon at the initiation of oncogenesis, which is
not only caused by mutations in the replication licensing
pathway. On the other hand, at least in some cases,
oncogene-induced deregulation of DNA replication occurs
through modulating the activities of replication licensing
factors. For instance, expression of the oncogenic cyclin D1
mutation P287A led to increased Cdt1 expression and DNA
re-replication. The human esophageal carcinoma-derived cell
lines TE3 and TE7, harboring the cyclin D1-P287A mutation
express Cdt1 5 –6-fold higher than the control cell lines due to disruption of the Cul4 – Ddb1– Cdt2-mediated Cdt1 degradation
pathway (Aggarwal et al., 2007). It was also shown that overexpression of oncogenes H-RasV12 caused increases in Cdc6
protein levels (Di Micco et al., 2006). Therefore, DNA
re-replication is an integral aspect of tumorigenesis, which activates cellular anti-tumor barriers while inducing genome instability. Subsequent accumulation of mutations in DNA damage
checkpoint pathways would abrogate the cellular anti-tumor barriers and lead to oncogenesis.
Unanswered questions
DNA re-replication is tightly associated with tumorigenesis, which
highlights the importance to understand the mechanisms underlying the prevention of re-replication. Despite recent significant
progress, many questions remain unanswered. For instance,
endoreduplication occurs during normal development to form
megakarocytes and trophoblastic cells (Zybina and Zybina,
2005; Deutsch and Tomer, 2006). It is not clear how cells distinguish this normal developmental endoreduplication from
abnormal re-replication and how checkpoints respond to them
differently. In addition, activation of ATR-mediated S-phase
checkpoint suppresses re-replication, but initial DNA
re-replication is needed to activate this checkpoint. It is expected
that low levels of DNA re-replication and short stretches of
re-replicated DNA are present in cells despite the suppression
of re-replication by the checkpoints. The mechanisms involved
in removing these initial re-replicated DNA to maintain genome
stability are not clear. Furthermore, loss of the licensing control
by Cdt1 overexpression or Geminin inactivation results in
20 | Journal of Molecular Cell Biology
chromosomal instability. It is necessary to elucidate the mechanisms of how DNA re-replication and its associated DNA lesions
lead to generation of specific chromosomal abnormalities commonly present in tumors, such as chromosomal translocation,
gene amplification and aneuploidy. Clarifying the interplay of
DNA re-replication control, checkpoint activation and repair of
re-replication-associated DNA lesion would be fundamental to
address these questions.
Acknowledgement
The authors thank the members of the Wu laboratory for
discussions.
Conflict of interest: none declared.
Funding
This work was supported by the NIH R01 Grant CA102361 and NIH
R01 Grant GM080677 to X.W., and the NIH Training Grant
DK007022-30 to L.T.
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