STRUCTURE AND FUNCTION OF HOMOLOGOUS RECOMBINATION ENZYMES

同源重组酶的结构和功能

• 批准号: 6997980
• 负责人: SCOTT W MORRICAL
• 金额: $ 19.55万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-03 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6997980
• 关键词: DNA binding protein; DNA damage; DNA repair; N glycosidase; affinity chromatography; binding sites; biochemistry; catalyst; crystallization; enzyme structure; fluorescence resonance energy transfer; gene targeting; genetic recombination; genetic screening; high throughput technology; ionizing radiation; recombinant proteins

Double-strand break repair (DSBR) by homologous recombination repairs double strand breaks produced by ionizing radiation and this DNA repair process is conserved from bacteriophage to humans. In all organisms, homologous DSBR requires the DNA strand exchange activity of an ortholog of the E. coli RecA protein. The RecA family is the most highly sequence-conserved family of DNA repair proteins, which exhibits strong conservation of global structure and function in promoting homologous recombination and DSBR transactions. Nevertheless, RecAs for which biochemical data are available exhibit surprising diversity in DNA binding properties, catalysis, and allosteric mechanisms, while others appear to be specialized recombination mediators. Does this diversity/specialization reflect different evolutionary paths towards RecA function? Also, to what extent does RecA biochemical diversity/specialization reflect the environmental demands placed on the DNA recombination and repair systems of a host organism? The work proposed in this Project intends to address these questions by studying the functional and structural diversity of RecA enzymes. In SPECIFIC AIM 1 we will compare biochemical and structural properties of divergent members of the RecA family. Target enzymes identified by computational methods will be cloned, expressed and purified. Using high throughput methods, DNA-binding and catalytic properties of each RecA ortholog will be determined. Variations in biochemical properties will be correlated with Phylogenetic and/or predicted structural variations within the RecA enzyme family. RecA orthologs representing distinct Phylogenetic and/or biochemical classes will be crystallized in the presence/absence of bound nucleotide, polynucleotide, and mediator protein ligands, and their high resolution X-ray structures will be determined. In SPECIFIC AIM 2 we will study the allosteric mechanisms of divergent RecA enzymes. The roles of key amino acid residues in the induction of the high-affinity ssDNA-binding state will be determined for selected RecA orthologs. The ability of cognate recombination mediator proteins (RMPs) to induce high-affinity RecA-ssDNA binding will also be examined in targeted systems.

通过电离辐射产生的同源重组维修双链断裂，这种DNA修复过程是从噬菌体到人类的保守的。在所有生物体中，同源DSBR都需要大肠杆菌RECA蛋白的直系同源物的DNA链交换活性。 RECA家族是最高度序列保存的DNA修复蛋白家族，它在促进同源重组和DSBR交易方面表现出强烈的全球结构和功能。然而，可获得生化数据的RECA在DNA结合特性，催化和变构机制方面表现出令人惊讶的多样性，而另一些则似乎是专门的重组介质。这种多样性/专业化是否反映了对RECA功能的不同进化途径？另外，RECA生化多样性/专业化在多大程度上反映出对宿主生物体的DNA重组和修复系统的环境需求？该项目中提出的工作旨在通过研究RECA酶的功能和结构多样性来解决这些问题。在特定目标1中，我们将比较RECA家族不同成员的生化和结构特性。通过计算方法鉴定的目标酶将被克隆，表达和纯化。使用高吞吐量方法，将确定每个RECA直系同源物的DNA结合和催化特性。生化特性的变化将与系统发育和/或 预测RECA酶家族中的结构变化。在存在/不存在结合的核苷酸，多核苷酸和介体蛋白配体的情况下，将确定代表不同系统发育和/或生化类别的RECA直系同源物，并确定其高分辨率X射线结构。 在特定目标2中，我们将研究发散酶的变构机制。对于选定的RECA直系同源物，将确定关键氨基酸残基在诱导高亲和力ssDNA结合态的作用。同源重组介质蛋白（RMP）诱导高亲和力RECA-SSDNA结合的能力也将在靶向系统中进行检查。