Structures of priming and recombination complexes

描述(申请人提供)：DNA复制叉子的持续移动需要在滞后的链上启动以形成每个Okazaki片段，以及引物和复制DNA聚合酶的协调作用。单链DNA(SsDNA)噬菌体G4(G4ori)的复制起点是一种简单的DNA复制启动系统，它只需要大肠杆菌引物酶(Dna G)和单链DNA结合蛋白(SSB)。G4ori由三个茎环组成，位于起始三核苷酸5‘-CTG-3’的5‘侧，当与SSB四聚体饱和结合时，将形成dNAG Primase可识别的独特结构。然后，这三个结构域协同结合G4ori起始点，合成引物RNA。G4ori(G4ori278)的278个核苷酸片段与两个dNAG引物和四个SSB四聚体一起，可以形成一个最小的活性引物复合体，适合于结构研究。复制叉的持续运动还需要DNA损伤部位的重组修复，而recf重组途径的recFOR复合体是复制和重组的介导者。这些复制和重组复合体的结构信息将有助于我们理解复制机制的功能。为此，我们提出了三组实验：1)确定SSB-G4ori278的双组分复合体和SSB与LacZ基因片段的复合体的X射线结构。这些结构将有助于我们理解功能G4ori的结构形成，以及SSB与单链DNA结合的模式。2)确定了Primase-SSB-G4ori278三元复合体的X射线结构，揭示了Primase-G4ori相互作用以及Primase-SSB相互作用的细节。3)确定RECR的X射线结构，并结晶其他蛋白质以及RECF途径中的复合体。获得这些蛋白质和复合体的结构信息将是理解重组修复的重要一步。

DESCRIPTION (provided by applicant): The continuous movement of the DNA replication fork requires priming at the lagging strand to make each Okazaki fragment, as well as a coordinated action of primases and the replicative DNA polymerase. The replication origin of the single-stranded DNA (ssDNA) bacteriophage G4 (G4ori) is a simple system of priming for DNA replication; it needs only the E. coli primase (dnaG) and the single-stranded DNA binding protein (SSB). G4ori consists of three stem-loops located immediately on the 5' side of the initiation trinucleotide 5'-CTG-3' and, upon saturated binding by SSB tetramers, will form a unique structure recognizable by dnaG primase. The three domains of the modular primase then bind the G4ori initiation site cooperatively to synthesize the primer RNA. A 278 nucleotide fragment of G4ori (G4ori278), together with two dnaG primases and four SSB tetramers, can form a minimal active priming complex suitable for structural investigations. The continuous movement of the replication fork also requires recombinational repair at sites of DNA damage and recFOR complexes of the recF recombination pathway are mediators of replication and recombination. Structural information of these replication and recombination complexes will help us understand the function of the replication machineries. Toward this goal, we propose three sets of experiments: 1) To determine the X-ray structures of the two-component complex of SSB-G4ori278 and a complex of SSB with a fragment of the lacZ gene. These structures will help us understand the structural formation of the functional G4ori, as well as SSB's modes of ssDNA-binding. 2) To determine the X-ray structure of the three-component complex of primase-SSB-G4ori278, which will reveal details of the primase- G4ori interaction, as well as the primase-SSB interaction. 3) To determine the X-ray structure of recR and to crystallize other proteins, as well as complexes in the recF pathway. Obtaining the structural information of these proteins and complexes will serve as an important step in understanding the recombinational repair.