Campuses:

Supercoiled Minicircle DNA to Probe Topoisomerase-DNA Interactions

Sunday, September 16, 2007 - 3:15pm - 4:15pm
EE/CS 3-180
Lynn Zechiedrich (Baylor College of Medicine)
Joint work with Jonathan M. Fogg, Daniel J. Catanese, Jr.,
Department of Molecular Virology & Microbiology, Baylor
College of Medicine, Houston, TX USA.

DNA topoisomerases are essential enzymes that impact nearly
every aspect of
DNA metabolism by transiently breaking and passing DNA.
Studies of
topoisomerases, indeed any protein that acts on DNA, have been
severely
limited by the lack of experimentally tractable supercoiled
DNA. We
developed a method to produce milligram quantities of DNA
minicircles (bp) with defined supercoiling (Fogg et al. 2006 J. Phys.:
Condens. Matter
,
18, S145). With these physiologically relevant substrates, we
have measured,
for the first time, the influence of supercoiling on a type-2
topoisomerase.
The importance of the development of supercoiled minicircles is
obvious from
initial data. Human topoisomerase IIα (hTopoIIα) binds linear,
nicked and
relaxed (ΔLk = 0) 339 bp minicircles with a
KD ~ 100 pM, which
agrees with
the previous data for linear DNA. We have determined that
hTopoIIα binds ΔLk
= -1, -2, -4 and -6 minicircles ~ 100-fold more tightly,
KD ~ 1 pM. There is no significant difference in binding over a wide range of
negative supercoiling from ΔLk = -1 to
ΔLk = -6. We have also studied hTopoIIα
binding to positively supercoiled DNA. The KD ~ 1 pM for
hTopoIIα binding to ΔLk = +2 and +3 was the same as that measured for
ΔLk = -2. Although it
is well-documented that type-2 enzymes preferentially relax (+)
over (-) supercoiled DNA, this difference is not accounted for in
binding. Therefore, the preference for (+) supercoils must be manifested in a later
kinetic step. The koff for hTopoIIα from nicked, linear, or relaxed
minicircle DNA was as fast as we could measure. The
kon, calculated from koff
and KD values, for all Lks was diffusion limited. However, the
koff for ΔLk = -2
minicircle is at least 5,000-fold slower! The extraordinary differences in
koff between ΔLk = 0 and
ΔLk = -2 likely reflect key regulatory features of
supercoiled DNA in cells as the ΔLk = -2 has the same
supercoiling state as is isolated from cultures of logarithmically growing cells.
Minicircle substrates provide a unique insight into the local DNA
structure of supercoiled DNA and how this is recognized and manipulated by
topoisomerases. Topoisomerases are major targets for
anti-cancer and anti-bacterial drugs. An improved understanding of how these
enzymes work may lead to rational structure-based approaches for the design
of new treatments.

This work was funded by National Institutes of Health (NIH)
Grant RO1
AI054830 (to LZ). DJC was funded by a training fellowship from
the Keck
Center Pharmacoinformatics Training Program of the Gulf Coast
Consortia, NIH
Grant T90 070109. JMF was funded by the Program in Mathematics
and
Molecular Biology.