Saturday, March 10, 2012

Topoisomerase in the Literature

Need to unwind?

Topoisomerase relieves the tension in DNA strands caused during replication by the helicase's procession along the helix toward a confining binding protein. It does so by cleaving and rejoining one strand of the DNA helix. Cleavage is a trans-esterification reaction in which the phosphodiester bond of DNA is attacked by a tyrosine-723, resulting in the formation of a DNA-(3′-phosphotyrosyl)-enzyme intermediate and the expulsion of the 5′-OH nucleotide and the rest of its strand. During strand rejoining, the 5′-OH end attacks the intermediate to reform a phosphodiester bond and expel the tyrosine.
Berit O Krogh1 and Stewart Shuman. Catalytic Mechanism of Topoisomerase IB. Molecular Cell. 5(6): 1035-1041, 1 June 2000.

DNA can never resist Topo's positive core

Topoisomerase I promote the relaxation of DNA superhelix tension. It does not require any metal co-factors. No ATP is consumed in the reaction. A highly positive core provides an attractive binding pocket for DNA. Two subdomains form a "cap" to the enzyme, which contacts the other sub-domains at only two lips, effectively forming a clamp around DNA.

Electron density map of covalent complex near active site. This shows the 3-phosphate-tyrosine bond between Tyr-723 and the broken strand of DNA.

A) Colored subdomains of the enzyme. B) View of enzyme down pore, with domains colored as in A. Central pore offers a highly positive binding region for DNA. The "cap" of the enzyme contacts core subdomain III at two lips (residues 367-369 of subdomain I and residues 497-499 of subdomain III).

 Matthew R. Redinbo,* Lance Stewart, Peter Kuhn, James J. Champoux, Wim G. J. Holt. Crystal Structures of Human Topoisomerase I in Covalent and Noncovalent Complees with DNA. Science 279 (5356). 6 March, 1998.1504-1513.

Cancer treatments target Topoisomerase 
Topoisomerase IB is overexpressed in mamalian tumor cells because of their increased replication. Camptothecin (CPT) and its derivatives, FDA approved cancer drugs, stabilize the topoisomerase-DNA covalent intermediate, preventing the 5'-OH of the free DNA strand from attacking and re-forming a phosphodiester bond with its partner. During S Phase, the replication forks collide with the topoisomerase-DNA complex and convert the single-strand breakage into a double strand breakage, and eventually apoptosis is signaled. CPT was originally isolated from the bark and stem of the Chinese tree Camptotheca acuminata, and it is presumed that its linear structure stabilizes the intermediate.
CPT- wikipedia
Rotated +1 Nucleoside model for Top1 interaction with dsDNA and 10-OH CPT. CPT is the spherically represented structure. Cyan blue is DNA, and yellow and red indicate enzymatic residues.

Laco, Gary S. Evaluation of Two Models for Human Topoisomerase I Interaction with dsDNA and Camptothecin Derivatives.  Public library of sciences. 6(8). 2011.

1 comment: