Coli DNA Topoisomerase I
David Kysela and
DNA topoisomerases are a class of enzymes involved
in the regulation of DNA supercoiling.
Type I topoisomerases change the degree of supercoiling of DNA by causing
single-strand breaks and re-ligation, whereas type II topoisomerases (such
as bacterial gyrase) cause double-strand breaks. The different roles of
DNA topoisomerase I and II may indicate an opposing pair of roles in the
regulation of DNA supercoiling. Both activities are especially crucial
during DNA transcription and replication, when the DNA helix must be unwound
to allow proper function of large enzymatic machinery, and topoisomerases
have indeed been shown to maintain both transcription and replication.
Recently, new topoisomerases of both types I and II (classified as topoisomerase
III and IV) have been discovered. These topoisomerases may indicate even
more roles for topoisomerases, with some topoisomerase III enzymes implicated
in regulation of recombination events, and topoisomerase IV implicated
in the process of segregating newly replicated chromosomes.
The focus of this tutorial is E. coli DNA topoisomerase I, responsible
primarily for the relaxation of negative supercoils. Topoisomerase I has
also been implicated in knotting and unknotting DNA and in linking complementary
rings of single-stranded DNA into double-stranded rings. The intact holoenzyme
is a 97K protein with three Zn(II) atoms in tertacysteine motifs near its
carboxy-terminus. Topoisomerase I appears to reverse supercoiling by transiently
breaking a segment of single-stranded DNA, passing an intact single- or
double-stranded strand of DNA through the gate, then rejoining the broken
segment. To understand the function of this mechanism, a supercoil may
be thought of as a knot on a string where the string is fixed at both ends,
and so the knot may not simply be untied. Instead, the gating mechanism
appears to open, untie, and reseal the "knotted" DNA precisely where it
The 67K N-terminal fragment that is the subject of this tutorial is
capable of this gating/passage activity, but not of directly relaxing negative
supercoils. Other domains include a zinc-binding domain implicated in the
process of relaxing DNA supercoils and a 14K C-terminal DNA-binding domain.
Topoisomerase I has several unusual features. Unlike type II topoisomerases,
topoisomerase I does not require ATP hydrolysis to catalyze the complex
topological rearrangements of DNA for which it is responsible. Whereas
most enzymes involved in complex rearrangements of DNA are oligomeric,
topoisomerase I appears to be a fully functional monomer.
The 67K N-terminal fragment of topoisomerase I is a single polypeptide
with extensive secondary structure <