2008 Scientific Report

VARI | 2008
Research Interests
We are studying how cells accurately replicate their DNA, a process that begins at specific DNA sequences termed replication
origins. Various genome-wide approaches have identified from 320 to 420 possible replication origins in budding yeast,
the model organism we study. In G1 phase, each origin assembles approximately 40 polypeptides in a temporally defined
order, culminating in the initiation of DNA replication at the G1/S phase boundary. The first stage of this process is called
pre-replicative complex assembly and requires the origin recognition complex (ORC), Cdc6p, and Cdt1p. ORC directly binds
to DNA and then recruits Cdt1p and Cdc6p during early G1 phase. These three proteins cooperate to load the MCM DNA
helicase at origins in an ATP-dependent reaction. Cyclin-dependent kinases and the Cdc7p-Dbf4p kinase then catalyze
the association of additional proteins with the MCM helicase, ultimately causing the initiation of bi-directional DNA synthesis
(Figure 1). In our lab we are studying how Cdc6p-ATP functions to load the MCM helicase within a chromatin context in
budding yeast. We also are studying the Cdc7p-Dbf4p kinase in both yeast and human cells.
We previously discovered that deletion of SIR2, encoding a histone deacetylase, rescued the temperature sensitivity of several
mutants that were defective in pre-RC assembly, including a cdc6-4 mutant. We screened the replication origins on chromosomes
III and VI to identify those origins that were inhibited by SIR2 and identified five SIR2-sensitive origins: ARS305, ARS315,
ARS317, ARS603, and ARS606. We determined the detailed structure of two origins on chromosome III and found that these
origins contain inhibitory elements distal to the ORC binding site. By utilizing data from another group that mapped stably
positioned nucleosomes on chromosome III, we found that these inhibitory elements were positioned within stably bound nucleosomes.
Furthermore, the positioned nucleosomes were very close to or overlapping the site of pre-RC assembly. Origins
that were not inhibited by SIR2 did not have nearby nucleosomes in this region. Since genetically, SIR2 inhibited origins through
the inhibitory element, we suggest that the acetylation state of this nucleosome affects pre-RC assembly. We do not know
whether Sir2p is acting directly at these inhibitory sites or indirectly by regulating another gene, but we put forth the following
model to explain our findings (Figure 2). Because pre-RC assembly occurs on naked DNA and because nucleosomes within
the origin inhibit pre-RC assembly, some origins may exist within a nucleosome environment that is not optimal for pre-RC
assembly. In those cases, either a particular modification of a nearby nucleosome or a protein that binds to the nucleosome
in a SIR2-dependent manner inhibits pre-RC assembly. Since Sir2p was previously thought to only act at very specific heterochromatic
regions in the genome, we are interested to discover exactly how Sir2p acts at these euchromatic sites.
Figure 1
Figure 1. The stages of replication initiation. In the first stage, a multi-subunit complex called the prereplicative
complex (pre-RC) is assembled at replication origins. This complex consists of ORC, Cdt1p, Cdc6p,
and the MCM helicase. In the second stage, Cdc7p-Dbf4p and Cdk kinases activate the MCM helicase,
resulting in origin unwinding and the association of DNA polymerases at the origin.
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