Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

THE INITIATION AND COMPLETION OF DNA REPLICATION IN CHROMOSOMES
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It seems that the order in which replication origins are activated depends, in
part, on the chromatin structure in which the origins reside. We saw in Chapter
4 that heterochromatin is a particularly condensed state of chromatin, while
euchromatin, where most transcription occurs, has a less condensed conformation.
Heterochromatin tends to be replicated very late in S phase, suggesting that
the timing of replication is related to the packing of the DNA in chromatin.
Once initiated, however, replication forks seem to move at comparable rates
throughout S phase, so the extent of chromosome condensation seems to influence
the time at which replication forks are initiated, rather than their speed once
formed.
A Large Multisubunit Complex Binds to Eukaryotic Origins of
Replication
Having seen that a eukaryotic chromosome is replicated using many origins of
replication, each of which “fires” at a characteristic time in S phase of the cell
cycle, we turn to the nature of these origins of replication. We saw earlier in this
chapter that replication origins have been precisely defined in bacteria as specific
DNA sequences that attract initiator proteins, which then assemble the DNA replication
machinery. We shall see that this is the case for the single-cell budding
yeast S. cerevisiae, but it appears not to be strictly true for most other eukaryotes.
For budding yeast, the location of every origin of replication on each chromosome
has been determined. The particular chromosome shown in Figure 5–30—
chromosome III from S. cerevisiae—is one of the smallest chromosomes known,
with a length less than 1/100 that of a typical human chromosome. Its major origins
are spaced an average of 30,000 nucleotide pairs apart, but only a subset of
these origins is used by a given cell. Nonetheless, this chromosome can be replicated
in about 15 minutes.
The minimal DNA sequence required for directing DNA replication initiation
in S. cerevisiae has been determined by taking a segment of DNA that spans an
origin of replication and testing smaller and smaller DNA fragments for their ability
to function as origins. Most DNA sequences that can serve as an origin of replication
are found to contain (1) a binding site for a large, multisubunit initiator
protein called ORC, for origin recognition complex; (2) a stretch of DNA that is
rich in As and Ts and therefore easy to melt; and (3) at least one binding site for
proteins that facilitate ORC binding, probably by adjusting chromatin structure.
In bacteria, once the initiator protein is properly bound to the single origin
of replication, the assembly of the replication forks seems to follow more or less
automatically. In eukaryotes, the situation is significantly different because of a
profound problem eukaryotes have in replicating chromosomes: with so many
places to begin replication, how is the process regulated to ensure that all the DNA
is copied once and only once?
The answer lies in the sequential manner in which the replicative helicase is
first loaded onto origins and is then activated to initiate DNA replication. This
matter is discussed in detail in Chapter 17, where we consider the machinery that
underlies the cell-division cycle. In brief, during G 1 phase, the replicative helicases
are loaded onto DNA next to ORC to create a prereplicative complex. Then,
upon passage from G 1 phase to S phase, specialized protein kinases come into
play to activate the helicases. The resulting opening of the double helix allows the
loading of the remaining replication proteins, including the DNA polymerases.
CHROMOSOME III
telomere
origins of replication
centromere
0 100
200
nucleotide pairs (thousands)
telomere
300
Figure 5–30 The origins of DNA
replication on chromosome III of the
yeast S. cerevisiae. This chromosome,
one of the smallest eukaryotic
chromosomes known, carries a total of
180 genes. As indicated, it contains 18
replication origins, although they are used
with different frequencies. Those in red
are typically used in less than 10% of cell
divisions, while those in green are used
about 90% of the time.