Springer, Encyclopedic Reference Of Cancer (2001) Ocr 7.0 Lotb.pdf

772 Replication Licensing System
RLF-B/Cdt1 acts to promote the loading of
the RLF-M complex onto DNA, but thereafter
does not appear to be required for the maintenance
of the licensed state. Its precise molecular
activity is currently unknown. RLF-B/Cdt1
is under tight cell cycle control and this regulation
is likely to be important in preventing rereplication
of DNA in a single cell cycle.
The Origin Recognition Complex (ORC)
consists of 6 polypeptides termed Orc1, 2, 3,
4, 5 and 6. Binding of ORC to DNA appears necessary
for that place to be defined as a replication
origin. In addition to its role in establishing
replication origins, ORC also appears to be involved
in establishing repressive chromatin
states at specific sites on DNA.
The binding of Cdc6 to chromatin is dependent
on the presence of ORC. It is required for
the origin to become licensed but is not for the
maintenance of the licensed state once licensing
has occurred. In mammalian cells, Cdc6
is exported from the nucleus at the end of
G1 and does not regain access to the chromatin
until the end of the subsequent mitosis. In
yeast, Cdc6 is degraded at the end of G1. These
features are likely to contribute to mechanisms
that prevent re-licensing of DNA once S phase
has started.
Regulation
Since re-replication of segments of chromosomal
DNA is likely to cause very significant genetic
changes to the cell, it is obviously crucial
for eukaryotic cells to prevent this from happening.
In order for the licensing system to operate
properly and ensure that no DNA re-replicates,
its activity must completely cease before
S phase starts (Fig. 2). Recent work suggests
that cells may possess multiple redundant
mechanisms for ensuring this. One important
consideration is that the components required
to load the MCM/P1 proteins onto DNA (ORC,
Cdc6, RLF-B/Cdt1) are not required for the
continued binding of the MCM/P1 proteins.
Therefore in principle, licensing can be prevented
by inhibiting the activity of either
ORC, Cdc6 or RLF-B/Cdt1/Cdt1, whilst the licensed
state is maintained. Although it is currently
not possible to describe in molecular de-
tail how licensing is prevented late in the cell
cycle, three important themes have emerged:
regulation by changes in subcellular localisation,
regulation by cyclin-dependent kinases
and regulation by geminin.
Early experiments that characterised the replication
licensing system showed that in order
for replicated nuclei from the G2 phase of the
cell cycle to undergo a further round of DNA
replication, they had to undergo a transient
permeabilization of their nuclear envelope.
This suggested that re-replication of chromosomal
DNA was regulated at least in part by compartmentalization
of regulatory components
between the nucleus and cytoplasm. Recent
work has confirmed this idea, but has shown
that different organisms actually regulate different
components of the licensing system by
this mechanism. In mammalian cells, the
Cdc6 protein, which is required on the DNA
for licensing to occur, is nuclear in late mitosis
and G1 and is exported from the nucleus during
S phase and G2. The lack of Cdc6 in the nucleus
late in the cell cycle is likely to play an important
role in preventing the re-licensing (and
hence re-replication) of the DNA. A different
version of this story is seen in the yeast Saccharomyces
cerevisiae, where the MCM/P1 proteins,
rather than Cdc6, are present in the nucleus
only in late mitosis and early G1 and are
exported from the nucleus at later stages of the
cell cycle. Physically separating essential licensing
components from their DNA substrate by
nuclear export would seem to be a powerful
way of preventing the inappropriate licensing
of replicated DNA.
Another important aspect of the regulation
of the replication licensing system involves cyclin-dependent
kinases (CDK). These kinases
promote the major transitions of the eukaryotic
cell division cycle, including entry into S phase
(SPF) and entry into mitosis (MPF) (Fig. 2).
During late mitosis and early G1, however,
CDK activity is low and this is the period
when replication licensing can occur. Research
in a number of different experimental systems
have shown that CDKs, either directly or indirectly,
can inhibit origin licensing. The nuclear
exclusion of yeast MCM/P1 proteins and mammalian
Cdc6 appears to be promoted by high