STRUCTURAL STUDIES OF DNA REPLICATION INITIATION

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。DNA复制叉的连续运动需要在滞后链上引发以产生每个冈崎片段，以及引发酶和复制DNA聚合酶的协调作用。单链DNA噬菌体G4（G4 ori）的复制起点是一个简单的DNA引发系统，它只需要E.大肠杆菌引发酶（dnaG）和单链DNA结合蛋白（SSB）。G4 ori可能由位于起始三核苷酸5-CTG-3的5侧的三个茎环组成，并且在被SSB四聚体饱和结合后，将形成可被dnaG引发酶识别的独特结构。然后，模块引发酶的三个结构域协同结合G4 ori起始位点以合成引物RNA。G4 ori的单链278个核苷酸片段（G4 ori 278）与两个dnaG引物酶和四个SSB四聚体一起可以形成适合于结构研究的最小活性引发复合物。我们已经分离并获得了两种类型的配合物的晶体：SSB-G4 ori 278和primase-SSB-G4 ori 278。SSB-G4 ori 278复合物不含dnaG引发酶，而引发酶-SSB-G4 ori 278复合物存在引发酶。获得这些复合物的原子分辨率结构将回答许多与G4噬菌体DNA的复制起点以及SSB和引发酶的结构-功能相关的问题，例如复制起点的茎-环的整体结构是什么以及SSB分子如何与ssDNA相互作用以形成复制起点的独特结构。SSB-G4 ori 278结构也将揭示SSB如何与DNA相互作用。此外，存在引发酶的复合物的结构将显示引发酶如何识别复制起点以及引发酶和SSB的相互作用。获得primase-SSB-G4 ori复合物的结构将为未来DNA引发和复制的结构和生物化学研究打开许多大门。例如，它将使我们能够研究引物合成的过程！通过获得与引发酶合成中间体的复合物结构来合成。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 DNA priming; it needs only the E. coli primase (dnaG) and the single-stranded DNA binding protein (SSB). G4ori consists possibly 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 single-stranded 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. We have isolated and obtained crystals of two types of complexes: SSB-G4ori278 and primase-SSB-G4ori278. The SSB-G4ori278 complex does not contain dnaG primase, while the primase-SSB-G4ori278 complex has primase present. Obtaining atomic resolution structures of these complexes will answer many functionally relevant questions on the replication origin of G4 phage DNA and the structure-function of SSB and primase, such as what is the overall structure of the stem-loops of the replication origin and how do the SSB molecules interaction with the ssDNA to form the unique structure of the replication origin. The SSB-G4ori278 structure will also shed light on how SSB interacts with DNA. Furthermore, the structure of the complex with the primase present will show how the primase recognize the replication origin and the interaction of primase and SSB. Obtaining the structure of the primase-SSB-G4ori complex will open many doors for future structural and biochemical studies of DNA priming and replication. For example, it will enable us to study the process of primer synth! esis by obtaining the complex structures of with the primase synthesis intermediates.