| dc.description.abstract |
<p>The accurate and timely replication of eukaryotic DNA during S-phase is of critical
importance for the cell and for the inheritance of genetic information. Missteps in
the replication program can activate cell cycle checkpoints or, worse, trigger the
genomic instability and aneuploidy associated with diseases such as cancer. Eukaryotic
DNA replication initiates asynchronously from hundreds to tens of thousands of replication
origins spread across the genome. The origins are acted upon independently, but patterns
emerge in the form of large-scale replication timing domains. Each of these origins
must be localized, and the activation time determined by a system of signals that,
though they have yet to be fully understood, are not dependent on the primary DNA
sequence. This regulation of DNA replication has been shown to be extremely plastic,
changing to fit the needs of cells in development or effected by replication stress.
</p><p>We have investigated the role of chromatin in specifying the eukaryotic DNA
replication program. Chromatin elements, including histone variants, histone modifications
and nucleosome positioning, are an attractive candidate for DNA replication control,
as they are not specified fully by sequence, and they can be modified to fit the unique
needs of a cell without altering the DNA template. The origin recognition complex
(ORC) specifies replication origin location by binding the DNA of origins. The <italic>S.
cerevisiae</italic> ORC recognizes the ARS (autonomously replicating sequence) consensus
sequence (ACS), but only a subset of potential genomic sites are bound, suggesting
other chromosomal features influence ORC binding. Using high-throughput sequencing
to map ORC binding and nucleosome positioning, we show that yeast origins are characterized
by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences
are sufficient to maintain a nucleosome-free origin; however, ORC is required for
the precise positioning of nucleosomes flanking the origin. These findings identify
local nucleosomes as an important determinant for origin selection and function. Next,
we describe the <italic>D. melanogaster</italic> replication program in the context
of the chromatin and transcription landscape for multiple cell lines using data generated
by the modENCODE consortium. We find that while the cell lines exhibit similar replication
programs, there are numerous cell line-specific differences that correlate with changes
in the chromatin architecture. We identify chromatin features that are associated
with replication timing, early origin usage, and ORC binding. Primary sequence, activating
chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute
in an additive manner to specify ORC-binding sites. We also generate accurate and
predictive models from the chromatin data to describe origin usage and strength between
cell lines. Multiple activating chromatin modifications contribute to the function
and relative strength of replication origins, suggesting that the chromatin environment
does not regulate origins of replication as a simple binary switch, but rather acts
as a tunable rheostat to regulate replication initiation events. </p><p>Taken together
our data and analyses imply that the chromatin contains sufficient information to
direct the DNA replication program.</p>
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