General methods for analysis of sequential "n-step'' kinetic mechanisms:: Application to single turnover kinetics of helicase-catalyzed DNA unwinding
General methods for analysis of sequential "n-step'' kinetic mechanisms:: Application to single turnover kinetics of helicase-catalyzed DNA unwinding
复制标题
DOI：
10.1016/s0006-3495(03)74648-7
复制
发表时间：
2003-10-01
影响因子：
3.4
通讯作者：
Lohman, TM
中科院分区：
文献类型：
--
作者：
Lucius, AL;Maluf, NK;Lohman, TM
解旋酶催化的 DNA 解旋通常使用“全部或无”测定来研究，该测定仅检测完全解旋 DNA 的最终产物。即使使用这些测定法，使用“n步”顺序机制对不同长度 L 的 DNA 双链体的 DNA 解旋时间过程进行定量分析，也可以揭示有关解旋反应中中间体数量和“动力学步长”m 的信息，m 定义为解旋循环中两个连续速率限制步骤之间解绕的碱基对的平均数量。使用“n 步”顺序机制的同时非线性最小二乘分析先前因无法在拟合算法中浮动“展开步骤”的数量（n 和 m）而受到限制。在这里，我们讨论单周转 DNA 解旋时间过程的行为，并描述克服这些问题的非线性最小二乘分析的新方法。当可以获得时，时间进程的解析表达式 f(ss)(t) 可以使用伽马和不完全伽马函数来编写。当无法获得解析表达式时，可以利用拉普拉斯逆变换的数值解来获得f(ss)(t)。两种方法都允许 n 和 m 为连续拟合参数。这些方法通常适用于沿着晶格易位或在产物形成之前需要重复一系列步骤的酶。
Helicase-catalyzed DNA unwinding is often studied using "all or none" assays that detect only the final product of fully unwound DNA. Even using these assays, quantitative analysis of DNA unwinding time courses for DNA duplexes of different lengths, L, using "n-step" sequential mechanisms, can reveal information about the number of intermediates in the unwinding reaction and the "kinetic step size", m, defined as the average number of basepairs unwound between two successive rate limiting steps in the unwinding cycle. Simultaneous nonlinear least-squares analysis using "n-step" sequential mechanisms has previously been limited by an inability to float the number of "unwinding steps", n, and m, in the fitting algorithm. Here we discuss the behavior of single turnover DNA unwinding time courses and describe novel methods for nonlinear least-squares analysis that overcome these problems. Analytic expressions for the time courses, f(ss)(t), when obtainable, can be written using gamma and incomplete gamma functions. When analytic expressions are not obtainable, the numerical solution of the inverse Laplace transform can be used to obtain f(ss)(t). Both methods allow n and m to be continuous fitting parameters. These approaches are generally applicable to enzymes that translocate along a lattice or require repetition of a series of steps before product formation.