20
Xenopus
to the extract, and replication does not require nuclear envelope assembly.
Another recent development includes using single-molecule
approaches in Xenopus egg extracts in the study replication
dynamics (Yardimci et al., 2012). In these elegant experiments, DNA templates are tethered to a substrate, and the
incorporation of nucleotides or the behavior of replication
proteins can be monitored, often in real time, with f uorescent nucleotides or proteins and f uorescence microscopy.
Using microf uidics, it is possible to block new origin f ring
by fowing in inhibitors, and the sequential addition of different nucleotides or labeled proteins can be accomplished.
In one example, fork progression was monitored in real time
by analyzing the movements of a f uorescent-PCNA binding protein on the immobilized template (Loveland et al.,
2012). In another, the impact of DNA replication on histone
dynamics was examined (Gruszka et al., 2020). Similar
approaches have been used to analyze the interaction of the
replication apparatus with chromatin-bound proteins, such
as the cohesin complex, or with DNA protein crosslinks
(Kanke et al., 2016; Sparks et al., 2019). These and other
single-molecule approaches to study chromosome biology
in Xenopus egg extracts have recently been well reviewed
elsewhere (Cameron and Yardimci, 2021).
Experiments in Xenopus egg extract have helped elucidate the mechanisms that link the completion of DNA replication to M phase entry. As Cyclin B accumulates in the
extract, MPF (Cdk1-Cyclin B) is prevented from activation
by a kinase called Myt1, a member of the Wee1 family of
kinases. Wee1, which was f rst identifed in f ssion yeast,
places inhibitory phosphorylations on Cdk1. The removal
of these phosphorylations by Cdc25 ensures mitotic entry
( Gautier et al., 1991 ; Kumagai and Dunphy, 1991 ). Cdc25
itself is controlled by periodic phosphorylation ( Izumi et al.,
1992 ). Incomplete DNA replication, stimulated by the addition of DNA polymerase inhibitors, prevents activation of
Cdc25, ensuring that MPF is not activated until DNA replication is complete ( Gabrielli et al., 1992 ). These kinds
of experiments have, in turn, led to many ground-breaking
experiments in the study of DNA damage signaling and
repair, reviewed elsewhere ( Hoogenboom et al., 2017 ).
2.7. ORIGINS AND TIMING
Work in bacterial and yeast models in the late 1970s suggested
that DNA replication originates at specifc DNA sequences. In
higher eukaryotes, the picture was less clear. Several groups
showed that DNA injected into frog eggs or egg extract would
spontaneously be replicated, even if the template contained
no eukaryotic DNA sequences (Harland and Laskey, 1980;
Mahbubani et al., 1992; Méchali and Kearsey, 1984). This led
to the model, which persists to this day, that replication origins were not sequence specifed in vertebrates.
Xenopus egg extracts provide a uniquely tractable system
with which to study the nature and distribution of vertebrate
replication origins. It was noted that the number of nuclei
added to extract could impact the rate of completion of DNA
replication, suggesting that titration of some soluble factor
in the extract could result in increased replicon size ( Walter
and Newport, 1997 ). DNA combing, another sort of singlemolecule experiment, was used to analyze the distribution of
active origins and helped to explain the eff ciency and speed
of genome duplication in the early embryo. In this technique,
DNA in egg extract is labeled by the addition of modif ed
nucleotides, which are then incorporated into nascent DNA.
The DNA is then isolated from the extract and spread on glass
slides where it can be probed for labeled nucleotides ( Blow
et al., 2001 ). The addition of a second labeled nucleotide to
the replication reaction at a later time point allows calculation of the distance between replication origins and the rate of
DNA replication ( Marheineke et al., 2009 ; Marheineke and
Hyrien, 2004 ). It also led to the observation that fork density, that is, the number of replication forks per unit length of
DNA, increased during replication progression through activation of later fring origins ( Herrick et al., 2000 ). Xenopus
egg extracts were also exploited to understand the nature of
replication origins in nuclei from other sources. For example,
the addition of Chinese hamster ovary (CHO) cell nuclei
to egg extract was used to demonstrate the time in the cell
cycle at which the somatic (CHO) cell origins were specif ed
( Dimitrova and Gilbert, 1999 ; Li et al., 2003 ). These experiments ultimately led to the development of the concept of replication timing ( Pope et al., 2013 ). Replication timing refers
to the phenomenon in which certain regions of the genome
reproducibly replicate earlier than others. Replication timing
is still an area of active investigation in many model systems.
The ability to manipulate the steps that lead to replication origin f ring have made Xenopus egg extracts uniquely
useful for studying events that depend on replication. For
example, the Cohesin complex, which establishes connections between sister chromatids during DNA replication, is
regulated by direct interaction of Cohesin regulators with
the replication machinery. Work in our lab and others has
shown dependencies and often direct interaction between
replication proteins and essential Cohesin regulators. The
Cohesin loader Scc2 binds to DDK (Cdc7-Dbf4), and the
Cohesin regulator Esco2 binds to chromatin in a licensingdependent manner and interacts with the PCNA sliding
clamp (Higashi et al., 2012; Lafont et al., 2010; Rankin et al.,
2005; Song et al., 2012; Takahashi et al., 2008, 2004). The
cohesion phenotypes of mitotic chromosomes can also be
analyzed by driving extract into M phase following DNA
replication (Silva and Rankin, 2018).
2.8. SUMMARY AND FUTURE DIRECTIONS
The eggs and embryos of Xenopus laevis have been used
for decades in foundational investigations of DNA replication and cell division. Through the collective work of many
dedicated researchers, we have a clearer understanding of
basic mechanisms driving the proliferation of cells. With
the ability to quickly and easily replicate DNA in vitro,
there is a promising future for research using Xenopus egg
extracts. Now that extracts can be compartmentalized using
Xenopus
to the extract, and replication does not require nuclear envelope assembly.
Another recent development includes using single-molecule
approaches in Xenopus egg extracts in the study replication
dynamics (Yardimci et al., 2012). In these elegant experiments, DNA templates are tethered to a substrate, and the
incorporation of nucleotides or the behavior of replication
proteins can be monitored, often in real time, with f uorescent nucleotides or proteins and f uorescence microscopy.
Using microf uidics, it is possible to block new origin f ring
by fowing in inhibitors, and the sequential addition of different nucleotides or labeled proteins can be accomplished.
In one example, fork progression was monitored in real time
by analyzing the movements of a f uorescent-PCNA binding protein on the immobilized template (Loveland et al.,
2012). In another, the impact of DNA replication on histone
dynamics was examined (Gruszka et al., 2020). Similar
approaches have been used to analyze the interaction of the
replication apparatus with chromatin-bound proteins, such
as the cohesin complex, or with DNA protein crosslinks
(Kanke et al., 2016; Sparks et al., 2019). These and other
single-molecule approaches to study chromosome biology
in Xenopus egg extracts have recently been well reviewed
elsewhere (Cameron and Yardimci, 2021).
Experiments in Xenopus egg extract have helped elucidate the mechanisms that link the completion of DNA replication to M phase entry. As Cyclin B accumulates in the
extract, MPF (Cdk1-Cyclin B) is prevented from activation
by a kinase called Myt1, a member of the Wee1 family of
kinases. Wee1, which was f rst identifed in f ssion yeast,
places inhibitory phosphorylations on Cdk1. The removal
of these phosphorylations by Cdc25 ensures mitotic entry
( Gautier et al., 1991 ; Kumagai and Dunphy, 1991 ). Cdc25
itself is controlled by periodic phosphorylation ( Izumi et al.,
1992 ). Incomplete DNA replication, stimulated by the addition of DNA polymerase inhibitors, prevents activation of
Cdc25, ensuring that MPF is not activated until DNA replication is complete ( Gabrielli et al., 1992 ). These kinds
of experiments have, in turn, led to many ground-breaking
experiments in the study of DNA damage signaling and
repair, reviewed elsewhere ( Hoogenboom et al., 2017 ).
2.7. ORIGINS AND TIMING
Work in bacterial and yeast models in the late 1970s suggested
that DNA replication originates at specifc DNA sequences. In
higher eukaryotes, the picture was less clear. Several groups
showed that DNA injected into frog eggs or egg extract would
spontaneously be replicated, even if the template contained
no eukaryotic DNA sequences (Harland and Laskey, 1980;
Mahbubani et al., 1992; Méchali and Kearsey, 1984). This led
to the model, which persists to this day, that replication origins were not sequence specifed in vertebrates.
Xenopus egg extracts provide a uniquely tractable system
with which to study the nature and distribution of vertebrate
replication origins. It was noted that the number of nuclei
added to extract could impact the rate of completion of DNA
replication, suggesting that titration of some soluble factor
in the extract could result in increased replicon size ( Walter
and Newport, 1997 ). DNA combing, another sort of singlemolecule experiment, was used to analyze the distribution of
active origins and helped to explain the eff ciency and speed
of genome duplication in the early embryo. In this technique,
DNA in egg extract is labeled by the addition of modif ed
nucleotides, which are then incorporated into nascent DNA.
The DNA is then isolated from the extract and spread on glass
slides where it can be probed for labeled nucleotides ( Blow
et al., 2001 ). The addition of a second labeled nucleotide to
the replication reaction at a later time point allows calculation of the distance between replication origins and the rate of
DNA replication ( Marheineke et al., 2009 ; Marheineke and
Hyrien, 2004 ). It also led to the observation that fork density, that is, the number of replication forks per unit length of
DNA, increased during replication progression through activation of later fring origins ( Herrick et al., 2000 ). Xenopus
egg extracts were also exploited to understand the nature of
replication origins in nuclei from other sources. For example,
the addition of Chinese hamster ovary (CHO) cell nuclei
to egg extract was used to demonstrate the time in the cell
cycle at which the somatic (CHO) cell origins were specif ed
( Dimitrova and Gilbert, 1999 ; Li et al., 2003 ). These experiments ultimately led to the development of the concept of replication timing ( Pope et al., 2013 ). Replication timing refers
to the phenomenon in which certain regions of the genome
reproducibly replicate earlier than others. Replication timing
is still an area of active investigation in many model systems.
The ability to manipulate the steps that lead to replication origin f ring have made Xenopus egg extracts uniquely
useful for studying events that depend on replication. For
example, the Cohesin complex, which establishes connections between sister chromatids during DNA replication, is
regulated by direct interaction of Cohesin regulators with
the replication machinery. Work in our lab and others has
shown dependencies and often direct interaction between
replication proteins and essential Cohesin regulators. The
Cohesin loader Scc2 binds to DDK (Cdc7-Dbf4), and the
Cohesin regulator Esco2 binds to chromatin in a licensingdependent manner and interacts with the PCNA sliding
clamp (Higashi et al., 2012; Lafont et al., 2010; Rankin et al.,
2005; Song et al., 2012; Takahashi et al., 2008, 2004). The
cohesion phenotypes of mitotic chromosomes can also be
analyzed by driving extract into M phase following DNA
replication (Silva and Rankin, 2018).
2.8. SUMMARY AND FUTURE DIRECTIONS
The eggs and embryos of Xenopus laevis have been used
for decades in foundational investigations of DNA replication and cell division. Through the collective work of many
dedicated researchers, we have a clearer understanding of
basic mechanisms driving the proliferation of cells. With
the ability to quickly and easily replicate DNA in vitro,
there is a promising future for research using Xenopus egg
extracts. Now that extracts can be compartmentalized using