The alpha4 residues of human DNA topoisomerase IIalpha function in enzymatic activity and anticancer drug sensitivity.
We introduced a series of Pro substitutions within and near the alpha4 helix, a part of the breakage/rejoining region, in human DNA topoisomerase IIalpha, 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 alpha4 helix residues are involved in the enzymatic activity and drug sensitivity of human DNA topoisomerase IIalpha, via interaction with cleaved DNA.
Suda
Namiko
N
Department of Endocrine Surgery, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan.
Ito
Yasutomo
Y
Imai
Tsuneo
T
Kikumori
Toyone
T
Kikuchi
Akihiko
A
Nishiyama
Yukihiro
Y
Yoshida
Shonen
S
Suzuki
Motoshi
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000502:physiology; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents / Q000494:pharmacology*; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D005047:Etoposide / Q000494:pharmacology; D008969:Molecular Sequence Data; D000075223:Poly-ADP-Ribose Binding Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D015003:Yeasts / Q000166:cytology / Q000187:drug effects
D000596:Amino Acids; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D005047:Etoposide; D004317:Doxorubicin; D004250:DNA Topoisomerases, Type II; C000618811:TOP2A protein, human