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J Biol Chem, Vol. 275, Issue 20, 15246-15253, May 19, 2000
From the Eukaryotic DNA topoisomerase I (Top1p) catalyzes
changes in DNA topology and is the cellular target of camptothecin.
Recent reports of enzyme structure highlight the importance of
conserved amino acids N-terminal to the active site tyrosine and the
involvement of Asn-726 in mediating Top1p sensitivity to camptothecin.
To investigate the contribution of this residue to enzyme catalysis, we
evaluated the effect of substituting His, Asp, or Ser for Asn-726 on
yeast Top1p. Top1N726S and Top1N726D mutant proteins were resistant to
camptothecin, although the Ser mutant was distinguished by a lack of
detectable changes in activity. Thus, a basic residue immediately
N-terminal to the active site tyrosine is required for camptothecin
cytotoxicity. However, replacing Asn-726 with Asp or His interfered
with distinct aspects of the catalytic cycle, resulting in cell
lethality. In contrast to camptothecin, which inhibits enzyme-catalyzed
religation of DNA, the His substituent enhanced the rate of DNA
scission, whereas the Asp mutation diminished the enzyme binding of
DNA. Yet, these effects on enzyme catalysis were not mutually exclusive
as the His mutant was hypersensitive to camptothecin. These results
suggest distinct mechanisms of poisoning DNA topoisomerase I may be
explored in the development of antitumor agents capable of targeting
different aspects of the Top1p catalytic cycle.
Substitutions of Asn-726 in the Active Site of Yeast DNA
Topoisomerase I Define Novel Mechanisms of Stabilizing the Covalent
Enzyme-DNA Intermediate*
,§,§
Department of Biochemistry and Molecular
Pharmacology, Thomas Jefferson University, Philadelphia,
Pennsylvania 19107, § Department of Molecular Pharmacology,
St. Jude Children's Research Hospital, Memphis Tennessee 38105, and
¶ Laboratory of Molecular Pharmacology, Division of Basic
Sciences, NCI, National Institutes of Health,
Bethesda, Maryland 20892-4255
*
This work was supported in part by National Institutes of
Health Grant CA58755 (to M.-A. B.), training Grants CA09137 and CA09346 (to J. R. V.), the CA21675 Cancer Center Grant, and
the American Lebanese Syrian Associated Charities.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. Molecular
Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Tel.: 901-495-2315; Fax: 901-521-1668;
E-mail: Mary-Ann.Bjornsti@stjude.org.
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