The Endoreduplication Cell Cycle: Regulation and Function 77
phase- and G2/M-phase-specific factors, and cyclin-dependent kinase (CDK)
activity.
2.1
The Control of Replication Origin Activation
Chromatin in eukaryotic cells contains an estimated 103 – 105 origins of repli-
cation, which typically are located 30 – 100 kb apart (Blow and Dutta 2005).
These origins have to be activated or “fired” in a highly coordinated fashion
to ensure complete and exact genome replication, with no refiring within one
S-phase. Early cell fusion studies with mammalian cells demonstrated that G1
nuclei can undergo DNA synthesis, whereas G2 nuclei cannot (Rao and John-
son 1970). It is now widely accepted that G1 nuclei can proceed to S-phase
because their chromatin is “licensed” for replication (Blow and Dutta 2005),
whereas G2 chromatin is not. The nature of this license resides in chromo-
somal replication origins that become primed during late M-phase and G1
for DNA synthesis. This process involves the stepwise loading of several pro-
teins, such as ORC, CDC6, CDT1, and the MCM2-7 complex (Sabelli et al.
1998). A drop in CDK activity, which occurs in late mitosis, is essential for
the assembly of this complex, and results in a so-called prereplicative complex
(pre-RC). Pre-RCs are essentially licensed origins that can be fired after a rise
in CDK activity, as occurs at the G1/S-phase transition.
Firing an origin of DNA replication involves localized unwinding of the
double-stranded DNA helix, and the recruitment of DNA replication enzymes
at the replication fork. The firing of replication origins results in a simultan-
eous loss of license, as many of the key licensing proteins become excluded
from chromatin and downregulated during S-phase by a range of CDK-driven
mechanisms, which include specific inhibitors, nuclear export, and prote-
olysis. This downregulation ensures that origins are prevented from being
relicensed and fired again during S-phase, and thus only one round of DNA
synthesis occurs during one cell cycle. This M-phase-dependent “window of
opportunity” for origin licensing, therefore, provides a crucial coupling be-
tween DNA synthesis and mitosis, which enables maintenance of genome
integrity through generations of cells. In molecular terms, the anaphase-
promoting complex/cyclosome (APC/C) ubiquitin ligase plays a key role
during mitosis in the degradation of specific proteins that would interfere
with pre-RC formation (Nakayama and Nakayama 2006) (see below). Dur-
ing endoreduplication, however, one or more rounds of DNA synthesis occur
without intervening mitoses, suggesting that deregulation of origin licensing
and firing might play an important role.
A considerable amount of information about the mechanisms that could
explain the repeated rounds of DNA replication typical of endoreduplication
may be provided by the study of the circumstances and causes of unscheduled
replication during G2 or M-phase. For example, inSaccharomyces cerevisiae,