There exists a complex interaction between the topological
structure of DNA and its metabolism. Processes such as replication, recombination,
transcription, and segregation are known to be remarkably sensitive to
DNA topology. However, the exact structural basis for these topological
effects remains undefined as the technical challenge of generating sufficient
amounts of small circular DNA makes has made it difficult to perform biophysical
analyses. I have recently discovered that topoisomerase IV is the exclusive
unknotting enzyme in vivo and that catalysis of this reaction is unaffected
by the negative supercoiling of the substrate. In contrast, the decatenating
activity of this enzyme is dramatically affected by the supercoiling of
the substrate. We intend to determine the structural parameter that differs
between DNA knots and catenanes using topoisomerase IV as a probe. Additionally,
we will undertake the first birefringence analyses of small, DNA of various
supercoiling densities, that we are able to generate in ample quantity
using an in vivo Int site-specific recombination system. As a result of
our proposed study we will more completely characterize topoisomerase IV
activity both in vitro and in vivo; delineate how the structure of knots
differs from that of catenanes; define how topoisomerases can recognize
the global topology of a DNA molecule; and perform the first birefringence
analysis on supercoiled DNA. |
