ALEXANDER ALEXANDROV |
Mechanism of DNA Unwinding by the Hexameric Helicases |
We are studying the mechanism of DNA unwinding by the
hexameric helicase SV-40 Large T-antigen and Bovine papillomavirus type
1 helicase E1. We have done extensive footprinting studies to probe the
contacts between T-antigen oligomer and an artificial replication fork.
Our studies have demonstrated that when bound to a fork Tantigen helicase
makes contacts with all three components of the replication fork: the 3' (polarity single strand) is protected over a distance of 45 nucleotides
starting from the ss/dsDNA junction; the 5' (displaced single strand) is
protected over a distance of 8ñ12 nucleotides counting from the ss/dsDNA
junction; up to 45 bps are protected in the duplex part of the DNA fork.
The imposed by the helicase oligomer is dependent on the presence and the
nature of the nucleotide cofactor bound to the helicase. We have tested
three different conditions: presence of AMP-PNP (a non-hydrolyzable ATP
analog), presence of ADP and absence of nucleotide cofactors. |
The footprinting results obtained under these conditions
represent snapshots of the different stages in the ATP turnover cycle of
the helicase. Taken together they allowed t~s to build a dynamic model
for the DNA unwinding cata]yzed by the T-antigen helicase. Our model provides
mechanistic explanation for the strand separating activity of the helicase
as well as the translocation activity. Our model predicts that T-antigen
is able to trans]ocate not only along ssDNA but also along duplex DNA.
This latter activity contributes to the processive unwinding activity of
T-antigen. |
The footprinting results were summarized as a manuscript,
which is in the process of preparation. |
We have also done some binding studies ó gel shift analysis
and filter binding to characterize quantitatively the binding of T-antigen
to ssDNA as well as the fork substrates. Our current results suggest that
the subunits of the T-antigen hexamer engage the ssDNA sequentially, reminiscent
of the way rho helicase binds its RNA substrate. These findings suggest
that hexameric RNA and DNA helicases share basic mechanistic similarities. |
