|
|
BENJAMIN TU |
Determination of Contact Interfaces of Yeast Topoisomerase II Through Use of a Novel Protein Footprinting Technique |
DNA topoisomerases are a class of fascinating enzymes that have become
the target of many interesting biochemical studies. They are enzymes that
disentangle DNA strands or duplexes in a cell, playing important roles
in DNA replication, transcription, chromosome condensation, and maintenance
of genome stability. Among the DNA topoisomerase subfamilies are the type
II enzymes. They are essential cellular enzymes that catalyze DNA topological
transformations by passing one double-stranded DNA segment through a transient
double stranded break in another. They have important roles in chromosome
segregation and altering DNA superhelicity. Very recently, eukaryotic topoisomerases
have become the target of many chemotherapeutical studies. DNA topoisomerase
11 is a cell cycle regulated enzyme; decatenating daughter DNA molecules
after replication. Inhibition of this process in rapidly dividing cancerous
cells may lead to their deaths. |
The most intriguing mechanistic aspect of DNA topoisomerases today are
the steps by which they move DNA strands or duplexes through one another.
The crystal structure of a large portion (92 kDa) of yeast topoisomerase
II has recently been solved. It reveals a pair of crescent-shaped monomcrs
which contact each other to form a ìVî-shaped dimer with a large central
hole. A molecular picture of the proposed mechanism can be described: The
G (gate)-segment of DNA is held in a pair of semicircular grooves near
the top of the ìVî-shaped enzyme. When the B'-subfragments are apart, a
second DNA duplex, termed the T (transport)-segment of DNA, can enter the
protein clamp through the N-terminal jaws at the top of the ìV.î Closure
of the terminal jaws, triggered by ATP binding, captures the T-segment
through the transiently cleaved and opened G-segment into a large cavity,
and is thus forced through the dimer interface at the bottom of the "V."
Of particular interest is the B-B' subfragment contact-interface suggested
by the crystal structure near the top of the ìV." It is hypothesized that
a dimerization of the B' subfragments may assist in the closure of the
N-terminal jaws and forcing of the T-segment through the G-segment. The
crystal structure and mechanistic hypotheses from previous studies strongly
suggest that the B'-B' interface is more than a simple crystallization-induced
contact. However, there is little published biochemical evidence that suggests
the B' subfragments interact with each other during dimerization and direct
transport of the T-sogmcnt DNA. The enzyme can be trapped in the form of
a closed clamp (closed N-terminal jaws) using a non-hydrolyzable ATP analogue.
Upon careful examination of the residues in this ìinterfaceî depicted in
the crystal structure, many experiments can be designed to determine whether
the B' subfragments actually interact with each other. Protein footprinting
and crosslinking experiments can be performed on selected amino acid residues
to determine the extent of the contacts in this interface. |
My research task has been to determine whether these B' subfragments
of yeast topoisomerase II contact each other during a cycle of DNA cleavage,
transport and religation through USC of a novel protein footprinting technique.
Protein footprinting allows identification of amino acid residues involved
in critical macromolecular contacts. The protein footprinting technique
I have been researching involves cysteine residues and their subsequent
modification using novel chemical methods. It provides the exciting opportunity
to perfect a new and useful technique for the study of protein protein
contacts. The project integrates molecular biological, biochemical, and
chemical approaches into answering fundamental questions on protein-protein
contacts and their roles in biological functions and catalytic mechanisms.
The project has also been extended to study the association and interaction
of the ATPase domains (not present in the solved crystal structure) at
the top of the enzyme through use of the same novel protein footprinting
technique. It is hopeful that knowledge of the movements and contacts of
these interfaces will soon elucidate the mechanism of this fascinating
enzyme and facilitate the design of topoisomerase-based anti-cancer drugs. |
My results thus far show that a significant contact interface forms
between the ATPase domains of a yeast topoisomerase II dimer during a cycle
of DNA cleavage, transport, and religation. A B' subfragment dimerization
interface during such a cycle appears to be somewhat less prominent; the
motion of the B' subfragments may be more complicated that originally expected.
I am currently in the process of reproducing and confirming my results.
I am also rigorously refining and evaluating the novel footprinting method. |
|
|
