454
in cleavage-stage embryos. Eventually, a limiting amount of this replication factor
leads to a delay in replication. While this is not DNA damage per se, it may be effec-
tively recognized as replication stress: ssDNA and ssDNA-dsDNA- binding proteins
like RPA, Rad17-RPC and 9-1-1 may have an opportunity to bind, leading to Chk1
activation. Supporting this possibility, a recent study identified replication factors that
could regulate cell cycle length during Xenopus cleavage divisions (Collart et al. 2013 ).
When these replication factors are abundant in the cleavage-stage environment, the
rapid origin firing may not allow binding of ATR and Chk1 activating factors to chro-
matin. However, the replication factors may become limiting at the MBT as the N:C
ratio increases, slowing replication and permitting ATR-Chk1 activation.
A second model is that the Chk1 adaptor protein Claspin is regulated by the N:C
ratio. Chk1 activation in somatic cells and Xenopus egg extracts depends on Claspin,
which recruits Chk1 to ATR for phosphorylation (Kumagai and Dunphy 2000 , 2003 ;
Chini and Chen 2003 ). Claspin is typically phosphorylated by ATR after replication
stress, but Xenopus embryos phosphorylate Claspin at low levels even in the absence of
ATR activity. However, Claspin phosphorylation is responsive to the N:C ratio: addi-
tion of sperm nuclei to Xenopus egg extracts to increase N:C ratios to MBT levels
resulted in Claspin phosphorylation in an ATR-independent manner. These data suggest
that a threshold N:C ratio may either activate a novel kinase or titrate a kinase inhibitor
to allow Claspin phosphorylation (Gotoh et al. 2011 ), in addition to the canonical phos-
phorylation of Claspin by ATR after MBT activation of the replication checkpoint.
Finally, a third hypothesis suggests that transcriptional activity itself can lead to
replication checkpoint activation, regardless of the products of transcription. In
Drosophila, binding of the replication protein RPA70 to DNA is tightly correlated
to DNA-binding of RNA polymerase II, which increases gradually throughout the
cleavage stages in preparation for zygotic transcription at the MBT (Blythe and
Wieschaus 2015b). These data support the hypothesis that sites of transcriptionally
engaged DNA can be sources of replication stress that activate the replication
checkpoint and confer cell cycle remodeling.
9.2.5 Checkpoint Acquisition at the MBT
The molecular mechanisms that underlie checkpoint acquisition at the MBT are poorly
understood. The following section reviews what is known about DNA damage check-
point and SAC acquisition at the MBT, with an emphasis on the role of the N:C ratio.
9.2.5.1 DNA Damage Checkpoint Acquisition
The first hints towards elucidating checkpoint regulation came from studies using
Xenopus egg extracts. As mentioned above, addition of DNA replication inhibitors,
DNA damaging agents, or microtubule poisons had no effect on cell cycle
progression in control extracts (Dasso and Newport 1990 ; Kumagai et al. 1998 ).
M. Zhang et al.