ENZYMATIC MECHANISMS OF E COLI DNA HELICASES

DNA的双螺旋结构要求存在一种机制， 用于解旋螺旋以暴露ssDNA用作模板或反应 DNA复制、修复和重组的中间体。 的DNA 解旋酶通过利用能量解旋双链体DNA来提供这种机制 由NTP水解提供。 已经描述了十多个解旋酶 在细菌E.大肠杆菌中的作用;每一个在DNA代谢中的作用是目前 正在澄清。 这项研究计划的长期目标是 理解，在酶和分子方面，作用机制， E. coli DNA解旋酶II、IV和Rep蛋白。 第一 该提案的两个具体目标涉及以下结构-功能研究： DNA解旋酶II，uvrD基因产物。 高度保守的氨基酸 残基（在解旋酶类内）将被位点改变， 特异性诱变，并分析突变蛋白产物 生物化学和遗传学。 这种方法将提供新的数据 关于反应机制，并有望产生新的突变体， 测试ATP酶和解旋酶活性在切除修复中的作用， 甲基指导的错配修复和DNA复制。此外，半- 随机诱变程序将用作开始定义 可能存在于解旋酶II中的各种活性结构域。 第三 具体目标涉及遗传和生物化学研究， 阐明解旋酶IV在细胞中的作用。 努力将集中在一个 解旋酶IV在重组中的作用。 第四个具体目标是 Rep蛋白利用其解旋酶活性 取代DNA上的蛋白质。 这种活性在DNA中可能很重要 因为复制叉必须遇到蛋白质-DNA 在DNA合成过程中。 此外，这些研究 开始解决解旋酶如何处理蛋白质结合的 在解旋反应过程中。

The double helical structure of DNA mandates the existence of a mechanism for unwinding the helix to expose ssDNA for use as a template or reaction intermediate in DNA replication, repair, and recombination. The DNA helicases provide such a mechanism by unwinding duplex DNA using energy provided by NTP hydrolysis. More than ten helicases have been described in the bacterium E. coli; the role of each in DNA metabolism is currently being elucidated. The long-range goal of this research program is to understand, in enzymatic and molecular terms, the mechanism of action and cellular role of E. coli DNA helicases Il, IV and Rep protein. The first two specific aims of this proposal involve structure-function studies of DNA helicase Il, the uvrD gene product. Highly conserved amino acid residues (within the class of helicase enzymes) will be altered by site- specific mutagenesis, and the mutant protein product will be analyzed biochemically and genetically. This approach will provide new data regarding the reaction mechanism, and is expected to yield new mutants to test the role of both ATPase and helicase activity in excision repair, methyl-directed mismatch repair, and DNA replication. In addition, a semi- random mutagenesis procedure will be used as a tool to begin to define the various activity domains likely to be present in helicase II. The third specific aim involves genetic and biochemical studies designed to elucidate the role of helicase IV in the cell. Efforts will focus on a role for helicase IV in recombination. The fourth specific aim centers on the possibility that Rep protein utilizes its helicase activity to displace proteins bound on DNA. This activity may be important in DNA replication since the replication fork must encounter protein-DNA complexes during the course of DNA synthesis. In addition, these studies begin to address the larger issue of how helicases deal with protein-bound DNA during the course of an unwinding reaction.