STRUCTURE AND FUNCTION OF HOMOLOGOUS RECOMBINATION ENZYMES

双链断裂修复(DSBR)通过同源重组修复电离辐射引起的双链断裂，这种DNA修复过程从噬菌体到人类都是保守的。在所有生物体中，同源DSBR需要大肠杆菌RecA蛋白的同源DNA链交换活性。RecA家族是DNA修复蛋白中序列高度保守的家族，在促进同源重组和DSBR交易方面表现出强大的全球结构和功能保守性。然而，有生化数据的RecAs在DNA结合特性、催化和变构机制上表现出惊人的多样性，而其他的似乎是专门的重组介体。这种多样性/专业化是否反映了RecA功能的不同进化路径？此外，RecA生化多样性/专门化在多大程度上反映了寄主生物体DNA重组和修复系统的环境要求？本项目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。在特定的目标1中，我们将比较RecA家族不同成员的生化和结构特性。通过计算方法确定的靶酶将被克隆、表达和纯化。使用高通量方法，将确定每个RecA直系物的DNA结合和催化性质。生化特性的变化将与系统发育和/或 预测了RecA酶家族的结构变异。代表不同系统发育和/或生化类别的RecA同源基因将在有/没有结合核苷酸、多核苷酸和介体蛋白配体的情况下结晶，并将确定它们的高分辨X射线结构。 在特定的AIM 2中，我们将研究不同RecA酶的变构机制。对于选定的RecA同源基因，将确定关键氨基酸残基在诱导高亲和力单链DNA结合状态中的作用。同源重组介体蛋白(RMPs)诱导高亲和力RecA-ssDNA结合的能力也将在目标系统中进行检测。

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.