406
Cell Nucleus Biogenesis, Structure and Function
optimal efFciency. These active centers
are also spatially structured through their
association with the nucleoskeleton. The
modular structure of replicon clusters also
provides a mechanism for replication to
spread in a predetermined way from one
bank of replicons to the next until the
S-phase is complete.
Aspects of crosstalk between nuclear
structure and function are of particular in-
terest when considering how the S-phase
program might be established. The im-
portance of nuclear space in setting up
the S-phase program is most evident in
S. cerevisiae
, in which the subnuclear po-
sition of replication origins in early G1
phase of the cell cycle determines whether
origins are used either early or late in
S-phase. In mammalian cells, the early
replicons are selected during the Frst one-
third of G1 phase of the cell cycle, prior
to the commitment to engage the cell cy-
cle. Two critical events appear to deFne
the replication program and these are be-
lieved to depend on the complex interplay
of chromosome structure, chromatin orga-
nization, and perhaps even global nuclear
architecture. Replicons that are activated
at the onset of the S-phase and most prob-
ably the downstream replication program
are established during the Frst 2 h of G1
phase of the cell cycle. This ‘‘temporal
decision point’’ coincides with the period
when chromosomal territories reestablish
their interphase structure and transcrip-
tional potential. The process of deFning
an S-phase program is distinct from the
process that establishes active origins of
DNA replication. This second process also
occurs in early G1 phase in mammalian
cells and is thought to involve the binding
of the origin recognition complex (ORC)
to particular chromosomal sites.
In human cells, the assembly of repli-
cation factories begins late in the G1
phase of the cell cycle and appears to take
about 15 min. The cell cycle machinery
is responsible for the cues that initiate
both the assembly process and initiation
of DNA synthesis. The replication ma-
chinery is recruited to appropriate DNA
foci – in mammalian cells only about 10%
of possible origins are activated at the
onset of the S-phase – through an asso-
ciation with ORCs on the DNA. All of the
components needed for synthesis are re-
cruited to the factories as a consequence
of protein–protein interaction. ±or many
of the replication components, factory-
targeting sequences have been deFned. As
many replicons are replicated in each fac-
tory (early S-phase DNA foci contain
5
replicons and later ones many more) the
structure of the complex dictates that chro-
matin must move into the active center,
which itself is static. This view can be con-
Frmed using G±P-PCNA to analyze the
spatial dynamics of replication sites in liv-
ing cells. This demonstrates that the foci,
once assembled, tend to be spatially stable,
with the PCNA (±ig. 1) molecules that are
assembled into the synthetic complex dur-
ing initiation remaining at the active site
until synthesis is complete. Once replica-
tion at a site is complete, the active center
will disassemble. Often a new active cen-
ter is then seen to assemble
de novo
,a
t
an adjacent site. This principle underlies
a proposed domino effect for a replication
program, in which local changes in chro-
matin architecture dictate how replication
proceeds until the S-phase is complete. In
mammalian cells, S-phase lasts for about
10 h, while DNA synthesis from a typ-
ical origin will continue for about 1 h.
As S-phase proceeds, the genome is re-
produced in a predictable order so that
deFned chromosomal regions are dupli-
cated at particular times during early, mid-
or late S-phase (±ig. 6).
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