UNWINDING AND TRANSLOCATION OF DNA BY HELICASES

DNA解旋酶几乎是DNA代谢的每一个方面所必需的，包括复制、修复、重组和转录。全面了解这些基本的生化过程需要了解DNA解旋酶的酶机制。这项研究的目标是使用我们开发的新方法，比以前更详细地研究解旋酶催化的DNA解绕。我们将研究两种病毒解旋酶作为广泛功能范围活动的代表。来自SV40病毒的SV40 T抗原是一种复制解旋酶，其功能为双六聚体，并以高度进程的方式解绕DNA。来自T4噬菌体的Dda参与复制起始和重组，以单体或二聚体的形式发挥作用，并以分布方式解开DNA。由于测量这些过程的方法不充分，DNA解绕和易位尚未得到很好的理解。我们已经开发了一种DNA解卷的预稳态分析，其中观察到的爆发幅度将作为酶和底物浓度的函数进行评估。该分析将作为活性位点滴定，并将有价值的解绕与ATP水解。两种方法将用于测量在单个催化循环中解开的碱基对的数量或“步长”：1)独特碱基对位置的解开速率将通过使用高锰酸钾对新形成的单链DNA进行化学修饰来确定；2)荧光光谱方法将通过将荧光核苷酸类似物结合到DNA底物的特定位点来测量独特位置的解开速率。ssDNA上的易位将通过测量与寡核苷酸末端结合的蛋白质的解旋酶催化位移的速率来研究。为了确定单链DNA上的运动方向是否存在偏差，将蛋白质块放置在寡核苷酸的两端。dsDNA在解绕反应中的功能意义将通过化学修饰的DNA底物来探测，以确定在反应周期中解旋酶与双链相互作用的程度。本文采用的新方法将以定量的方式定义DNA解旋酶的生化机制，这将有助于评估解旋酶在DNA代谢和疾病中的作用。

DNA helicases are required for virtually every aspect of DNA metabolism, including replication, repair, recombination and transcription. A comprehensive understanding of these essential biochemical processes requires knowledge of the enzymatic mechanisms of DNA helicases. The goal of this research is to use new methods that we have developed to investigate helicase-catalyzed DNA unwinding in greater detail than has previously been possible. We will study two viral helicases as representatives of a broad functional range of activities. SV40 T antigen, from the SV40 virus, is a replicative helicase that functions as a double hexamer and unwinds DNA in a highly processive manner. Dda, from bacteriophage T4, is involved in replication initiation and recombination, functions as a monomer or dimer, and unwinds DNA in a distributive manner. DNA unwinding and translocation are not well understood, due to inadequate methods to measure these processes. We have developed a pre-steady state assay for DNA unwinding in which the observed burst amplitude will be evaluated as a function of enzyme and substrate concentration. This assay will serve as an active site titration and will be valuable for relating unwinding to ATP hydrolysis. Two methods will be used to measure the number of base pairs unwound in a single catalytic cycle or "step size": 1) Unwinding rates at unique base pair positions will be determined by chemical modification of newly formed single-stranded DNA using potassium permanganate, and 2) A fluorescence spectroscopic method will be utilized to measure unwinding rates at unique positions by incorporating fluorescent nucleotide analogs into specific sites in DNA substrates. Translocation on ssDNA will be investigated by measuring the rate of helicase-catalyzed displacement of proteins bound to the ends of oligonucleotides. To determine whether there exists a bias in direction of movement on single-stranded DNA, the protein blocks will be placed on either end of the oligonucleotide. The functional significance of dsDNA during the unwinding reaction will be probed using chemically modified DNA substrates to determine the degree to which helicases interact with the duplex during the reaction cycle. The new methods utilized here will define the biochemical mechanisms of DNA helicases in a quantitative manner that will be useful for evaluating the role of helicases in DNA metabolism and disease.