MECHANISM OF ENERGY COUPLING BY DNA TOPOISOMERASE II

DNA 拓扑异构酶 II 的能量耦合机制

• 批准号: 6386047
• 负责人: JANET E. LINDSLEY
• 金额: $ 19.6万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386047
• 关键词: DNA; DNA gyrase; active sites; adenosine triphosphate; adenosinetriphosphatase; bioenergetics; biological transport; chemical kinetics; conformation; crosslink; enzyme mechanism; fluorescence spectrometry; protein binding

Type II DNA topoisomerases are essential enzymes that unlink intertwined chromosomes. By altering DNA topology they participate in chromosome segregation, condensation, replication, recombination and transcription. These enzymes are the targets of numerous clinically-used and trial-phase anticancer and antibiotic drugs, making the understanding of their mechanism medically relevant. The reaction these enzymes catalyze is the ATP-dependent transport of one segment of DNA through a transient break in a second segment of DNA. Recent pre-steady-state ATPase results indicate that the homodimeric topoisomerase II binds two ATP to initiate the reaction cycle, but hydrolyzes only one rapidly. The second ATP is hydrolyzed later in the reaction cycle, after a rate-determining step in the reaction. This strict sequential hydrolysis of the two ATP was completely unexpected and does not agree with any previous models for the mechanism of this enzyme. The goals of the present proposal are to further elucidate the ATPase mechanism, analyze the protein-DNA complex conformational changes during the reaction cycle, and determine when in this cycle DNA transport actually occurs. By combining all of these pre-steady-state and steady- state results, a more accurate mechanism for DNA topoisomerase II catalyzed reactions will be determined.

II 型 DNA 拓扑异构酶是解开缠绕的染色体的必需酶。 通过改变 DNA 拓扑结构，它们参与染色体分离、浓缩、复制、重组和转录。 这些酶是许多临床使用和试验阶段抗癌和抗生素药物的靶点，使得对其机制的理解具有医学意义。 这些酶催化的反应是一个 DNA 片段通过第二个 DNA 片段的短暂断裂进行 ATP 依赖性运输。 最近的前稳态 ATP 酶结果表明，同型二聚体拓扑异构酶 II 结合两个 ATP 以启动反应循环，但仅快速水解一个。 第二个 ATP 在反应循环的后期、反应中的速率决定步骤之后被水解。 两种 ATP 的这种严格顺序水解是完全出乎意料的，并且与该酶机制的任何先前模型都不相符。 本提案的目标是进一步阐明 ATP 酶机制，分析反应周期中蛋白质-DNA 复合物的构象变化，并确定在此周期中 DNA 转运实际发生的时间。 通过结合所有这些前稳态和稳态结果，将确定 DNA 拓扑异构酶 II 催化反应的更准确的机制。