Assignment of functional amino acids around the active site of human DNA topoisomerase IIalpha

Y Okada; Y Ito; A Kikuchi; Y Nimura; S Yoshida; M Suzuki

doi:10.1074/jbc.M003243200

Assignment of functional amino acids around the active site of human DNA topoisomerase IIalpha

J Biol Chem. 2000 Aug 11;275(32):24630-8. doi: 10.1074/jbc.M003243200.

Authors

Y Okada¹, Y Ito, A Kikuchi, Y Nimura, S Yoshida, M Suzuki

Affiliation

¹ First Department of Surgery and the Laboratories of Cancer Cell Biology and Medical Mycology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, 466-8550, Japan.

PMID: 10807924
DOI: 10.1074/jbc.M003243200

Abstract

An expression library for active site mutants of human topoisomerase IIalpha (TOP2alpha) was constructed by replacing the sequence encoding residues 793-808 with a randomized oligonucleotide cassette. This plasmid library was transformed into a temperature-sensitive yeast strain (top2-1), and viable transformants were selected at the restrictive temperature. Among the active TOP2alpha mutants, no substitution was allowed at Tyr(805), the 5' anchor of the cleaved DNA, and only conservative substitutions were allowed at Leu(794), Asp(797), Ala(801), and Arg(804). Thus, these 5 residues are critical for human TOP2alpha activity, and the remaining mutagenized residues are less critical for function. Using the x-ray crystal structure of yeast TOP2 as a structural model, it can be deduced that these 5 functionally important residues lie in a plane. One of the possible functions of this plane may be that it interacts with the DNA substrate upon catalysis. The side chains of Ser(803) and Lys(798), which confer drug resistance, lie adjacent to this plane.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Amino Acid Substitution
Animals
Antigens, Neoplasm
Base Sequence
Binding Sites
DNA Topoisomerases, Type II* / chemistry*
DNA Topoisomerases, Type II* / genetics
DNA Topoisomerases, Type II* / metabolism
DNA-Binding Proteins
Drosophila
Etoposide / pharmacology
Gene Library
Genetic Complementation Test
Humans
Isoenzymes / chemistry*
Isoenzymes / genetics
Isoenzymes / metabolism
Models, Molecular
Molecular Sequence Data
Mutagenesis, Insertional
Mutagenesis, Site-Directed
Oligodeoxyribonucleotides / chemistry
Poly-ADP-Ribose Binding Proteins
Protein Conformation
Recombinant Proteins / chemistry
Recombinant Proteins / metabolism
Saccharomyces cerevisiae / drug effects
Saccharomyces cerevisiae / enzymology
Saccharomyces cerevisiae / genetics
Sequence Alignment
Sequence Homology, Amino Acid
Yeasts

Substances

Antigens, Neoplasm
DNA-Binding Proteins
Isoenzymes
Oligodeoxyribonucleotides
Poly-ADP-Ribose Binding Proteins
Recombinant Proteins
Etoposide
DNA Topoisomerases, Type II
TOP2A protein, human