18
Xenopus
The mechanisms that ensure a single round of DNA
replication per cell cycle, and prevent re-replication, were
ultimately discovered to be complex and include some
redundancy. Licensing itself was defned as recruitment of
the Mini chromosome maintenance 2–7 (MCM2–7) protein
complex to replication origins, which were previously bound
by the Origin recognition complex (ORC) (Kubota et al.,
1997, 1995). The MCM hexamer is a DNA helicase that
ultimately unwinds the DNA helix allowing replication fork
progression. The Geminin protein, which had been identif ed
originally as an APC/C substrate destroyed in anaphase, was
shown to prevent MCM loading (McGarry and Kirschner,
1998). These studies suggested that destruction of Geminin
is in fact a part of the replication licensing system.
Although it was clear that Geminin was able to prevent
MCM loading, the mechanism was unknown. This mystery
was eventually solved with the discovery that Geminin binds
tightly to an essential replication licensing factor called Cdt1
( Maiorano et al., 2000 ; Wohlschlegel et al., 2000 ). Using the
Xenopus extract system, it was shown that degradation of
Geminin releases Cdt1, which can then bind to origins and
promote MCM binding. This in part explained the role of
the nuclear envelope: Geminin helped to retain Cdt1 in the
cytosol. Following degradation of Geminin, Cdt1 is released
to enter the nucleus, bind origins, and recruit the MCM complex. Unloading of MCM proteins following DNA replication helps to prevent re-replication.
Additional mechanisms were found to prevent DNA overreplication. The Cdt1 licensing factor itself was found to be
degraded during DNA replication, which led to the discovery of a previously unknown mechanism of protein turnover ( Arias and Walter, 2006 , 2005 ). Cdt1 degradation was
shown to result from its direct interaction with proliferating
cell nuclear antigen (PCNA), an essential replication protein.
PCNA acts as a sliding clamp to ensure the processivity of
DNA replication. Cdt1 interacts with PCNA through a PCNAinteracting protein (PIP) box, a conserved PCNA interaction
motif. The interaction between chromatin-bound PCNA and
Cdt1 through its PIP box was found to result in ubiquitination of Cdt1 by the Cul4 Cdt2 ubiquitin ligase, resulting in
degradation of Cdt1 by the proteasome. The PIP box of Cdt1
was ultimately shown to have unique properties that ensure its
modifcation by Cul4, and thus it was called a “PIP degron”
( Havens and Walter, 2009 ). Several other proteins were found
to have PIP degrons, ensuring their degradation during DNA
replication, while most PCNA-interacting proteins are not
affected by this mechanism. The destruction of Cdt1 during
DNA replication helps to ensure that origins do not become
relicensed, helping to prevent over-replication of DNA.
2.6. ORDERING EVENTS IN DNA
REPLICATION
The use of Xenopus egg extracts has been essential to our
understanding of the events that ensure controlled DNA
replication in vertebrates. The synchrony and speed with
which nuclei added to egg extracts undergo regulated DNA
replication has been a great advantage in assigning orders
of action of the various replication proteins. In a typical
experiment, antibodies against a particular protein of interest are generated and used to immune-deplete the protein
from the extract. The extract is then tested for replication
by the addition of nuclei or other DNA substrates. Rescue
experiments can easily be performed using recombinant
proteins expressed in bacterial, insect, or human cells. This
can be done with wild-type and mutant protein derivatives,
allowing mapping of essential functions to protein domains
or residues. At any time during progression through DNA
replication, chromatin can be isolated from the extract and
the proteins associated with the chromatin assessed, often
by immunoblot. It is also possible to program the extract to
express proteins from synthetic RNA added to the extract
or to add radiolabeled in vitro translated proteins to the
extract. These kinds of approaches have been used repeatedly to understand the order of binding and the interdependencies among a large number of replication proteins and
their regulators.
In addition to the protein recruitment steps that lead to
replication initiation, replication is also dependent on the
activities of several kinases. As with other replication initiation steps, mechanisms requiring kinase activities were
identifed using Xenopus egg extracts and supported the
fndings of parallel studies in other systems. Some kinases
were found to control the activity of replication proteins
directly; others were shown to provide feedback between the
cell cycle machinery and DNA replication control.
The change in MPF kinase activity during cell cycle transitions helps to orchestrate DNA replication. The binding of
the ORC to replication origins is inhibited by MPF, ensuring that ORC binding occurs only after exit from M phase
(Carpenter et al., 1996; Romanowski et al., 1996; Rowles
et al., 1996). Additionally, activation of the APC ubiquitin
ligase by MPF results in Cyclin destruction and exit from M
phase (and thus ORC binding) and also causes the destruction of the replication licensing inhibitor Geminin (McGarry
and Kirschner, 1998). Cell cycle-dependent destruction of
Geminin ensures that licensing occurs only once during the
cell cycle, during exit from M phase.
The initiation of DNA replication following replication
licensing is tightly controlled by two additional kinases:
Cdc7 and Cdk2. The Cdc7 kinase, previously identif ed in
fungal models, was shown to be essential for DNA replication. In Xenopus, Cdc7 interacts with one of two activating subunits: Drf1 in extracts and the early embryo and
Dbf4 later in development (Jares et al., 2004; Silva et al.,
2006; Takahashi and Walter, 2005). Cdc7, also called Dbf4dependent kinase (DDK) phosphorylates the MCM complex, which in turn leads to recruitment of a second kinase
called Cdc45 to the replication origin (Jares and Blow, 2000).
Cdc45 is localized to the site of local DNA unwinding with
the MCM complex during DNA replication and is essential
for loading of the GINS (go-ichi-ni-san) complex (Kubota
et al., 2003; Mimura et al., 2000; Pacek et al., 2006).
The GINS complex is required for both DNA replication
Xenopus
The mechanisms that ensure a single round of DNA
replication per cell cycle, and prevent re-replication, were
ultimately discovered to be complex and include some
redundancy. Licensing itself was defned as recruitment of
the Mini chromosome maintenance 2–7 (MCM2–7) protein
complex to replication origins, which were previously bound
by the Origin recognition complex (ORC) (Kubota et al.,
1997, 1995). The MCM hexamer is a DNA helicase that
ultimately unwinds the DNA helix allowing replication fork
progression. The Geminin protein, which had been identif ed
originally as an APC/C substrate destroyed in anaphase, was
shown to prevent MCM loading (McGarry and Kirschner,
1998). These studies suggested that destruction of Geminin
is in fact a part of the replication licensing system.
Although it was clear that Geminin was able to prevent
MCM loading, the mechanism was unknown. This mystery
was eventually solved with the discovery that Geminin binds
tightly to an essential replication licensing factor called Cdt1
( Maiorano et al., 2000 ; Wohlschlegel et al., 2000 ). Using the
Xenopus extract system, it was shown that degradation of
Geminin releases Cdt1, which can then bind to origins and
promote MCM binding. This in part explained the role of
the nuclear envelope: Geminin helped to retain Cdt1 in the
cytosol. Following degradation of Geminin, Cdt1 is released
to enter the nucleus, bind origins, and recruit the MCM complex. Unloading of MCM proteins following DNA replication helps to prevent re-replication.
Additional mechanisms were found to prevent DNA overreplication. The Cdt1 licensing factor itself was found to be
degraded during DNA replication, which led to the discovery of a previously unknown mechanism of protein turnover ( Arias and Walter, 2006 , 2005 ). Cdt1 degradation was
shown to result from its direct interaction with proliferating
cell nuclear antigen (PCNA), an essential replication protein.
PCNA acts as a sliding clamp to ensure the processivity of
DNA replication. Cdt1 interacts with PCNA through a PCNAinteracting protein (PIP) box, a conserved PCNA interaction
motif. The interaction between chromatin-bound PCNA and
Cdt1 through its PIP box was found to result in ubiquitination of Cdt1 by the Cul4 Cdt2 ubiquitin ligase, resulting in
degradation of Cdt1 by the proteasome. The PIP box of Cdt1
was ultimately shown to have unique properties that ensure its
modifcation by Cul4, and thus it was called a “PIP degron”
( Havens and Walter, 2009 ). Several other proteins were found
to have PIP degrons, ensuring their degradation during DNA
replication, while most PCNA-interacting proteins are not
affected by this mechanism. The destruction of Cdt1 during
DNA replication helps to ensure that origins do not become
relicensed, helping to prevent over-replication of DNA.
2.6. ORDERING EVENTS IN DNA
REPLICATION
The use of Xenopus egg extracts has been essential to our
understanding of the events that ensure controlled DNA
replication in vertebrates. The synchrony and speed with
which nuclei added to egg extracts undergo regulated DNA
replication has been a great advantage in assigning orders
of action of the various replication proteins. In a typical
experiment, antibodies against a particular protein of interest are generated and used to immune-deplete the protein
from the extract. The extract is then tested for replication
by the addition of nuclei or other DNA substrates. Rescue
experiments can easily be performed using recombinant
proteins expressed in bacterial, insect, or human cells. This
can be done with wild-type and mutant protein derivatives,
allowing mapping of essential functions to protein domains
or residues. At any time during progression through DNA
replication, chromatin can be isolated from the extract and
the proteins associated with the chromatin assessed, often
by immunoblot. It is also possible to program the extract to
express proteins from synthetic RNA added to the extract
or to add radiolabeled in vitro translated proteins to the
extract. These kinds of approaches have been used repeatedly to understand the order of binding and the interdependencies among a large number of replication proteins and
their regulators.
In addition to the protein recruitment steps that lead to
replication initiation, replication is also dependent on the
activities of several kinases. As with other replication initiation steps, mechanisms requiring kinase activities were
identifed using Xenopus egg extracts and supported the
fndings of parallel studies in other systems. Some kinases
were found to control the activity of replication proteins
directly; others were shown to provide feedback between the
cell cycle machinery and DNA replication control.
The change in MPF kinase activity during cell cycle transitions helps to orchestrate DNA replication. The binding of
the ORC to replication origins is inhibited by MPF, ensuring that ORC binding occurs only after exit from M phase
(Carpenter et al., 1996; Romanowski et al., 1996; Rowles
et al., 1996). Additionally, activation of the APC ubiquitin
ligase by MPF results in Cyclin destruction and exit from M
phase (and thus ORC binding) and also causes the destruction of the replication licensing inhibitor Geminin (McGarry
and Kirschner, 1998). Cell cycle-dependent destruction of
Geminin ensures that licensing occurs only once during the
cell cycle, during exit from M phase.
The initiation of DNA replication following replication
licensing is tightly controlled by two additional kinases:
Cdc7 and Cdk2. The Cdc7 kinase, previously identif ed in
fungal models, was shown to be essential for DNA replication. In Xenopus, Cdc7 interacts with one of two activating subunits: Drf1 in extracts and the early embryo and
Dbf4 later in development (Jares et al., 2004; Silva et al.,
2006; Takahashi and Walter, 2005). Cdc7, also called Dbf4dependent kinase (DDK) phosphorylates the MCM complex, which in turn leads to recruitment of a second kinase
called Cdc45 to the replication origin (Jares and Blow, 2000).
Cdc45 is localized to the site of local DNA unwinding with
the MCM complex during DNA replication and is essential
for loading of the GINS (go-ichi-ni-san) complex (Kubota
et al., 2003; Mimura et al., 2000; Pacek et al., 2006).
The GINS complex is required for both DNA replication