Nucleic Acids Research Advance Access originally published online on September 4, 2008
Nucleic Acids Research 2008 36(17):5635-5644; doi:10.1093/nar/gkn557
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2008, Vol. 36, No. 17 5635-5644
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
A single mutation in the 729 residue modulates human DNA topoisomerase IB DNA binding and drug resistance
1Department of Biology, University of Padova, Via U. Bassi 58/B, Padua 35131, 2Department of Biology, University of Rome Tor Vergata, Via Della Ricerca Scientifica, Rome 00133 and 3CASPUR Inter-University Consortium for the Application of Super-Computing for Universities and Research, Via dei Tizii 6, Rome 00185, Italy
*To whom correspondence should be addressed. Tel: +39 049 8276289; Fax: +39 049 8276300; Email: pietro.benedetti{at}unipd.it
Received June 10, 2008. Revised August 18, 2008. Accepted August 18, 2008.
Human DNA topoisomerase I (hTop1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of the antitumor drug camptothecin (CPT). The X-ray crystal structure of the enzyme covalently joined to DNA and bound to the CPT analog Topotecan suggests that there are two classes of mutations that can produce a CPT-resistant enzyme. The first class includes changes in residues that directly interact with the drug, whereas a second class alters interactions with the DNA and thereby destabilizes the drug binding site. The Thr729Ala, that is part of a hydrophobic pocket in the enzyme C-terminal domain, belongs to a third group of mutations that confer CPT resistance, but do not interact directly with the drug or the DNA. To understand the contribution of this residue in drug resistance, we have studied the effect on hTop1p catalysis and CPT sensitivity of four different substitutions in the Thr729 position (Thr729Ala, Thr729Glu, Thr729Lys and Thr729Pro). Tht729Glu and Thr729Lys mutants show severe CPT resistance and furthermore, Thr729Glu shows a remarkable defect in DNA binding. We postulate that the maintenance of the hydrophobic pocket integrity, where Thr729 is positioned, is crucial for drug sensitivity and DNA binding.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.