Binding of ATP to human DNA topoisomerase I resulting in an alteration of the conformation of the enzyme.
It has been known for some time that ATP inhibits the DNA relaxation activity of human DNA topoisomerase I. However, the underlying mechanism of this inhibitory effect remains largely unknown. Using filter binding assays, the binding of human DNA topoisomerase I to DNA was decreased in the presence of ATP. This result suggests that the inhibition of DNA relaxation activity of human DNA topoisomerase I by ATP is at the binding step rather than at the nicking or resealing step. DNA topoisomerase I cleavage assay further supports this notion. ATP-agarose binding and UV cross-linking assays also demonstrate that ATP directly and specifically binds human DNA topoisomerase I. To address whether the ATP binding results in conformational changes in human DNA topoisomerase I, various proteases were employed for detecting potential protein conformational changes. Our results indicated that the proteolytic susceptibilities of trypsin and chymotrypsin were altered in the presence of ATP. The result suggests that the conformation of human DNA topoisomerase I was altered upon ATP binding. In addition, the binding between ATP and human DNA topoisomerase I was also reduced by increasing concentrations of DNA. Our data suggests that human DNA topoisomerase I exhibits at least two incompatible conformations. One conformation is in the form of a topoisomerase I-ATP complex, which inhibits DNA relaxation activity of human DNA topoisomerase I, and the other, a topoisomerase I-DNA complex, which exerts DNA relaxation activity. Our studies identify the role of ATP in the regulation of human DNA topoisomerase I and provide a substantial implication of how human DNA topoisomerase I compromises its versatile functions.
Chen
H J
HJ
Institute of Biochemistry, School of Life Science, National Yang-Ming University, Taipei, Taiwan.
Hwang
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D004247:DNA / Q000378:metabolism / Q000494:pharmacology; D004264:DNA Topoisomerases, Type I / Q000187:drug effects / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D009690:Nucleic Acid Conformation; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects
D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D004264:DNA Topoisomerases, Type I