Published online 16 March 2004
Nucleic Acids Research, 2004, Vol. 32, No. 5 1767-1773
© 2004 Oxford University Press
The 
4 residues of human DNA topoisomerase II function in enzymatic activity and anticancer drug sensitivity
Department of Endocrine Surgery, 1 Divisions of Molecular Carcinogenesis and 2 Molecular Mycology and Medicine, Center for Neural Disease and Cancer and 3 Department of Virology, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan
*To whom correspondence should be addressed. Tel: +81 52 744 2456; Fax: +81 52 744 2457; Email: msuzuki{at}med.nagoya-u.ac.jp
Present address: Shonen Yoshida, Kaikoukai Rehabilitation Hospital, Amagun, Aichi 490-1405, Japan
We introduced a series of Pro substitutions within and near the 
4 helix, a part of the breakage/rejoining region, in human DNA topoisomerase II, and analyzed if this region is involved in determination of anti-cancer drug sensitivity in a temperature- sensitive yeast strain (top2-4 allele). Among the 19 mutants generated, H759P and N770P showed resistance to etoposide and doxorubicin at the non-permissive temperature, where cell growth depends on activity of the human enzyme. For these residues, mutants with an Ala substitution were further created, in which H759A also showed resistance to etoposide. H759P, H759A and N770P were expressed, purified and subjected to in vitro measurement of drug sensitivity. They generated lower amounts of the etoposide-induced cleavable complexes, and were also found to have lower decatenation activity than the wild-type. In the crystal structure, the yeast equivalent of His759 is found in the vicinity of the Arg713, a putative anchoring residue of the 3'-side of cleaved DNA strands. These results suggest that His759 and the other 4 helix residues are involved in the enzymatic activity and drug sensitivity of human DNA topoisomerase II, via interaction with cleaved DNA.