Specification of ORC activity by differential DNA affinity.
Madeleine A. Palacios-DeBeer, Ulrika Muller, Catherine A. Fox
Biomolecular Chemistry, U.W. Madison, 1300 University Ave, Madison, WI
53706-1532, United States
We present evidence that ORC's affinity for
its site within a silencer modulates its functions at HMR. The
HMR-E silencer binds the ORC with a remarkably high-affinity
in vitro compared to a large number of non-silencer replication
origins. This high-affinity is due to the putative B1 element at
HMR-E that with the ACS contains a near perfect match to the
'expanded' ORC binding site defined by the Newlon laboratory.
Interestingly, swap experiments indicate that the high-affinity ORC
binding site within HMR-E is sufficient to convert a weak
silencer and relatively strong replication origin into a strong silencer
and weak replication origin. The replication origin behavior is the same
regardless of SIR2 genotype. Thus replication origin efficiency
and silencer efficiency are inversely correlated. The precise sequence
of the B1 element is not important; rather a close match to the expanded
consensus site and high-affinity ORC-DNA interaction in vitro is
all that is necessary to convert a weak silencer to a strong silencer.
In addition, the strength of the 4 different HM silencers correlates
with their ORC binding affinity in vitro. We propose that a high-affinity ORC-DNA interaction promotes ORC's silencing function while
directly suppressing ORC's replication function. Mechanistic
investigations into how a high-affinity ORC-DNA interaction regulates
the balance between silencing and replication of HMR will be
presented.
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