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
261
Despite this, a human ORC that is very similar to the yeast ORC binds to origins
of replication and initiates DNA replication in humans. Many of the other
proteins that function in the initiation process in yeast likewise have central roles
in humans. It therefore seems likely that the yeast and human initiation mechanisms
are similar in outline, but chromatin structure, transcriptional activity, or
some property of the genome other than a specific DNA sequence has the central
role in attracting ORC and specifying mammalian origins of replication. These
ideas could also help to explain how a given mammalian cell chooses which of the
many possible origins to use when it replicates its genome and how this choice
could differ from cell to cell. Clearly, we have a great deal to discover about the
fundamental process of DNA replication initiation.
New Nucleosomes Are Assembled Behind the Replication Fork
Several additional aspects of DNA replication are specific to eukaryotes. As discussed
in Chapter 4, eukaryotic chromosomes are composed of roughly equal
mixtures of DNA and protein. Chromosome duplication therefore requires not
only the replication of DNA, but also the synthesis and assembly of new chromosomal
proteins onto the DNA behind each replication fork. Although we are
far from understanding this process in detail, we are beginning to learn how the
fundamental unit of chromatin packaging, the nucleosome, is duplicated. The cell
requires a large amount of new histone protein, approximately equal in mass to
the newly synthesized DNA, to make the new nucleosomes in each cell cycle. For
this reason, most eukaryotic organisms possess multiple copies of the gene for
each histone. Vertebrate cells, for example, have about 20 repeated gene sets, most
containing the genes that encode all five histones (H1, H2A, H2B, H3, and H4).
Unlike most proteins, which are made continuously, histones are synthesized
mainly in S phase, when the level of histone mRNA increases about fiftyfold as
a result of both increased transcription and decreased mRNA degradation. The
major histone mRNAs are degraded within minutes when DNA synthesis stops at
the end of S phase. The mechanism depends on special properties of the 3ʹ ends
of these mRNAs, as discussed in Chapter 7. In contrast, the histone proteins themselves
are remarkably stable and may survive for the entire life of a cell. The tight
linkage between DNA synthesis and histone synthesis appears to reflect a feedback
mechanism that monitors the level of free histone to ensure that the amount
of histone made exactly matches the amount of new DNA synthesized.
As a replication fork advances, it must pass through the parental nucleosomes.
In the cell, efficient replication requires chromatin remodeling complexes (discussed
in Chapter 4) to destabilize the DNA–histone interfaces. Aided by such
complexes, replication forks can transit even highly condensed chromatin efficiently.
As a replication fork passes through chromatin, the histones are transiently
displaced leaving about 600 nucleotide pairs of non-nucleosomal DNA in its
wake. The reestablishment of nucleosomes behind a moving fork occurs in an
intriguing way. When a nucleosome is traversed by a replication fork, the histone
octamer appears to be broken into an H3-H4 tetramer and two H2A-H2B dimers
(discussed in Chapter 4). The H3-H4 tetramer remains loosely associated with
DNA and is distributed at random to one or the other daughter duplex, but the
H2A-H2B dimers are released completely from DNA. Freshly made H3-H4 tetramers
are added to the newly synthesized DNA to fill in the “spaces,” and H2A-H2B
dimers—half of which are old and half new—are then added at random to complete
the nucleosomes (Figure 5–32). The formation of new nucleosomes behind
a replication fork has an important consequence for the process of DNA replication
itself. As DNA polymerase δ discontinuously synthesizes the lagging strand
(see pp. 253–254), the length of each Okazaki fragment is determined by the point
at which DNA polymerase δ is blocked by a newly formed nucleosome. This tight
coupling between nucleosome duplication and DNA replication explains why the
length of Okazaki fragments in eukaryotes (~200 nucleotides) is approximately
the same as the nucleosome repeat length.