Assembly and Activation of Enzyme-ssDNA Complexes

酶-ssDNA复合物的组装和激活

• 批准号: 8018659
• 负责人: SCOTT W MORRICAL
• 金额: $ 31.61万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8018659
• 关键词: ATP Hydrolysis; Antibiotics; Bacteriophage T4; Binding; Biochemical; Biochemistry; Cells; Communicable Diseases; Complex; DNA; DNA biosynthesis; DNA replication fork; Defect; Diagnosis; Enzyme Activation; Enzymes; Exhibits; Filament; Fluorescence; Genetic Recombination; Genome; Kinetics; Light; Link; Maintenance; Malignant Neoplasms; Mediator of activation protein; Methods; Modeling; Molecular Conformation; Mutagenesis; Organism; Pathway interactions; Polymerase; Prevention; Process; Property; Proteins; Public Health; Recruitment Activity; Research; SS DNA BP; Sedimentation process; Single-Stranded DNA; Spectrum Analysis; System; Testing; Thermodynamics; antitumor drug; cancer therapy; crosslink; fluorescence imaging; helicase; human disease; presynaptic; programs; protein protein interaction; recombinase; recombinational repair; single molecule; treatment strategy; tumor

DESCRIPTION (provided by applicant): The objective of this research program is to determine the mechanisms by which recombinase and helicase enzymes are assembled onto single-stranded DNA (ssDNA) in the bacteriophage T4 DNA replication/recombination system. We will study the assembly of the T4 UvsX recombinase into presynaptic filaments, and we will study the assembly of two different DNA helicases, Gp41 and Dda, onto ssDNA at replication forks and in recombination intermediates. All three enzymes must assemble onto ssDNA in the cell that is already covered with tightly bound Gp32, the T4 ssDNA-binding protein. UvsX and Gp41 both require the activity of a specific mediator protein, UvsY or Gp59, respectively, for proper assembly onto Gp32-ssDNA complexes, whereas Dda achieves the same effect through direct protein-protein interactions with Gp32. We will explore all three enzyme loading mechanisms using classical biochemical methods (kinetics, thermodynamics, fluorescence, sedimentation, crosslinking), singlemolecule approaches (fluorescence imaging, force spectroscopy), and mutagenesis. Our SPECIFIC AIMS are: (1) Determine the kinetic mechanism of UvsX-ssDNA presynaptic filament assembly and collapse. We will test a model in which UvsY protein selectively enhances filament nucleation, UvsX actively displaces gp32 from ssDNA, and filaments exhibit dynamic instability linked to ATP hydrolysis. (2) Determine how interactions of T4 Gp59 protein with replication fork DNA control helicase assembly and polymerase blockage. We will test a model in which cooperative binding of Gp32 to lagging-strand ssDNA converts Gp59 from a polymerase-blocking to a helicase-loading conformation that recruits Gp41 helicase to the replication fork. (3) Determine how interactions with Gp32 modulate the DNA helicase functions of T4 Dda protein. We will test a model in which Dda-Gp32 protein-protein interactions promote the oligomerization of Dda and enhance its DNA unwinding properties in both replication and recombination transactions. Understanding how helicase and recombinase enzymes are correctly assembled onto ssDNA is fundamental to understanding DNA replication, recombination, and repair mechanisms that are conserved in all organisms. There are clear links between errors in DNA replication/recombination/repair machineries and human disease states including cancer. Understanding how recombinase- and helicasessDNA complexes are correctly assembled and activated may therefore aid in the prevention, diagnosis, and treatment of cancer. PUBLIC HEALTH REVELANCE: Proper assembly of enzyme-ssDNA complexes is critical for genome replication and maintenance in all organisms. Defects in enzyme-ssDNA assembly processes are implicated in cancer and other human disease states. Enzyme-ssDNA assembly pathways are also potential targets for new classes of antibiotic and antitumor drugs. Our studies of enzyme-ssDNA assembly mechanisms may therefore shed light on how tumors develop and how infectious diseases progress, and may also suggest new treatment strategies.

描述（由申请人提供）：本研究计划的目的是确定重组酶和解旋酶在噬菌体T4 DNA复制/重组系统中组装到单链DNA（ssDNA）上的机制。我们将研究T4 UvsX重组酶组装成突触前纤维，我们将研究两种不同的DNA解旋酶Gp 41和Dda在复制叉和重组中间体上组装到ssDNA上。所有三种酶必须组装到细胞中的ssDNA上，细胞中已经被紧密结合的Gp 32（T4 ssDNA结合蛋白）覆盖。UvsX和Gp 41都需要一个特定的介体蛋白，UvsY或Gp 59的活性，分别为适当的组装到Gp 32-ssDNA复合物，而Dda通过直接与Gp 32的蛋白质-蛋白质相互作用达到相同的效果。我们将探索所有三种酶加载机制，使用经典的生化方法（动力学，热力学，荧光，沉降，交联），单分子方法（荧光成像，力谱），和诱变。我们的具体目标是：（1）确定UvsX-ssDNA突触前纤维组装和塌陷的动力学机制。我们将测试一个模型，其中UvsY蛋白选择性地增强丝成核，UvsX积极取代ssDNA的gp 32，和丝表现出动态不稳定性与ATP水解。(2)确定T4 Gp 59蛋白与复制叉DNA的相互作用如何控制解旋酶组装和聚合酶阻断。我们将测试一个模型，其中Gp 32滞后链ssDNA的合作结合Gp 59从聚合酶阻断转换为解旋酶加载构象，招募Gp 41解旋酶的复制叉。(3)确定与Gp 32的相互作用如何调节T4 Dda蛋白的DNA解旋酶功能。我们将测试一个模型，其中Dda-Gp 32蛋白质-蛋白质相互作用促进Dda的寡聚化并增强其在复制和重组交易中的DNA解旋特性。理解解旋酶和重组酶如何正确地组装到ssDNA上是理解DNA复制、重组和修复机制的基础，这些机制在所有生物体中都是保守的。DNA复制/重组/修复机制中的错误与包括癌症在内的人类疾病状态之间存在明确的联系。因此，了解重组酶和解旋酶DNA复合物如何正确组装和激活可能有助于预防，诊断和治疗癌症。 公共卫生：酶-ssDNA复合物的正确组装对于所有生物体的基因组复制和维持至关重要。酶-ssDNA组装过程中的缺陷与癌症和其他人类疾病状态有关。酶-ssDNA组装途径也是新型抗生素和抗肿瘤药物的潜在靶点。因此，我们对酶-ssDNA组装机制的研究可能揭示肿瘤如何发展和传染病如何进展，也可能提出新的治疗策略。