Assignment of functional amino acids around the active site of human DNA topoisomerase IIalpha.
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.
Okada
Y
Y
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.
Ito
Y
Y
Kikuchi
A
A
Nimura
Y
Y
Yoshida
S
S
Suzuki
M
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000951:Antigens, Neoplasm; D001483:Base Sequence; D001665:Binding Sites; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D004330:Drosophila; D005047:Etoposide / Q000494:pharmacology; D015723:Gene Library; D005816:Genetic Complementation Test; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D000075223:Poly-ADP-Ribose Binding Proteins; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015003:Yeasts
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D000075223:Poly-ADP-Ribose Binding Proteins; D011994:Recombinant Proteins; D005047:Etoposide; D004250:DNA Topoisomerases, Type II; C000618811:TOP2A protein, human