Mutation of GLY721 alters DNA topoisomerase I active site architecture and sensitivity to camptothecin

doi:10.1074/jbc.M705781200

HOME

QUICK SEARCH:

	Author:		Keyword(s):
Year:		Vol:		Page:

A more recent version of this article appeared on February 8, 2008

This Article

	Full Text (Accepted Manuscript)
	All Versions of this Article: 283/6/3305 most recent M705781200v1
	Alert me when this article is cited
	Alert me if a correction is posted

Services

	Email this article to a friend
	Similar articles in this journal
	Similar articles in PubMed
	Alert me to new issues of the journal
	Download to citation manager
	Request Permissions

Citing Articles

	Citing Articles via HighWire
	Citing Articles via Google Scholar

Google Scholar

	Articles by van der Merwe, M.
	Articles by Bjornsti, M.-A.
	Search for Related Content

PubMed

	PubMed Citation
	Articles by van der Merwe, M.
	Articles by Bjornsti, M.-A.

Social Bookmarking

	What's this?

Papers In Press, published online ahead of print December 4, 2007
J. Biol. Chem, 10.1074/jbc.M705781200
Submitted on July 13, 2007
Revised on November 7, 2007
Accepted on December 3, 2007

Mutation of GLY⁷²¹ alters DNA topoisomerase I active site architecture and sensitivity to camptothecin

Marié van der Merwe and Mary-Ann Bjornsti

Dept. of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105

Corresponding Author: Mary-Ann.Bjornsti{at}stjude.org

DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA via a concerted mechanism of DNA strand cleavage and religation. Top1p is the cellular target of the anticancer drug camptothecin (CPT), which reversibly stabilizes a covalent enzyme-DNA intermediate. Top1p clamps around duplex DNA, wherein the core and C-terminal domains are connected by extended a-helices (linker domain), which position the active site Tyr of the C-terminal domain within the catalytic pocket. The physical connection of the linker with the Top1p clamp as well as linker flexibility affect enzyme sensitivity to CPT. Crystallographic data that reveal a conserved Gly residue (located at the juncture between the linker and C-terminal domains) is at one end of a short a-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this a-helix extends to include Gly and the preceding Leu. We report that mutation of this conserved Gly in yeast Top1p alters enzyme sensitivity to CPT. Mutating Gly to Asp, Glu, Asn, Gln, Leu or Ala enhanced enzyme CPT sensitivity, with the acidic residues inducing the greatest increase in drug sensitivity in vivo and in vitro. By contrast, Val or Phe substituents rendered the enzyme CPT resistant. Mutation-induced alterations in enzyme architecture preceding the active site Tyr suggest these structural transitions modulate enzyme sensitivity to CPT, while enhancing the rate of DNA cleavage. We postulate that this conserved Gly residue provides a flexible hinge within the Top1p catalytic pocket to facilitate linker dynamics and the structural alterations that accompany drug binding of the covalent enzyme-DNA intermediate.

Add to CiteULike CiteULike Add to Complore Complore Add to Connotea Connotea Add to Del.icio.us Del.icio.us Add to Digg Digg Add to Reddit Reddit Add to Technorati Technorati What's this?

This article has been cited by other articles:

K. Palle, L. Pattarello, M. van der Merwe, C. Losasso, P. Benedetti, and M.-A. Bjornsti
Disulfide Cross-links Reveal Conserved Features of DNA Topoisomerase I Architecture and a Role for the N Terminus in Clamp Closure
J. Biol. Chem., October 10, 2008; 283(41): 27767 - 27775.
[Abstract] [Full Text] [PDF]

G. Chillemi, I. D'Annessa, P. Fiorani, C. Losasso, P. Benedetti, and A. Desideri
Thr729 in human topoisomerase I modulates anti-cancer drug resistance by altering protein domain communications as suggested by molecular dynamics simulations
Nucleic Acids Res., October 1, 2008; 36(17): 5645 - 5651.
[Abstract] [Full Text] [PDF]

C. Losasso, E. Cretaio, P. Fiorani, I. D'Annessa, G. Chillemi, and P. Benedetti
A single mutation in the 729 residue modulates human DNA topoisomerase IB DNA binding and drug resistance
Nucleic Acids Res., October 1, 2008; 36(17): 5635 - 5644.
[Abstract] [Full Text] [PDF]