Enzymatic Mechanisms of DNA Synthesis

描述（由申请人提供）：真核生物基因组由复制叉合成，其中一条链被制成短片段，称为冈崎片段，然后将其连接。每个片段以RNA引物起始，然后将DNA添加到大约100个核苷酸中。来自上游片段的聚合将下游片段上的起始RNA置换成一个瓣。皮瓣内切酶（FEN1）去除皮瓣，留下切口进行结扎。一些襟翼被认为需要额外的Dna2核酸酶/解旋酶切割。最近的证据也表明Pif1和Bloom （BLM）解旋酶也有作用。利用遗传信息为指导，我们开发了一个重建冈崎片段连接的生化系统。早期的结果表明，虽然一些皮瓣很容易被FEN1处理，但其他皮瓣变得很长，需要Dna2的作用。我们将测试Dna2是否促进长皮瓣的正确加工。Pif1突变抑制Dna2突变，表明Pif1产生了对Dna2的需求。BLM过表达抑制了Dna2突变，表明BLM消除了对Dna2的需求。这些蛋白质的功能关系将被确定。在RNA之后添加的DNA被认为有频繁的错配错误。我们将确定链置换合成是否进一步进入含有错配片段，并由Pif1辅助，以允许5'校对。FEN1和Dna2都位于皮瓣的5'末端，并移动到它们的卵裂点，但两者都不干扰对方的作用。我们将分析这两种蛋白质的运动，以确定这种协调是如何可能的。BLM是DNA连接酶i的强刺激物，两种蛋白质的物理和功能相互作用将被检查。长补片碱基切除修复（Long patch base excision repair, LP BER）与Okazaki片段加工非常相似，同样使用FEN1和DNA连接酶i。911检查点复合体、AP内切酶和PCNA都协调和刺激LP BER蛋白。我们将采用免疫沉淀、底物结合和活性刺激实验来区分这三种蛋白在LP BER通路中的促进作用。我们将扩大初步结果表明DNA损伤诱导911和LP BER蛋白之间更有效的相互作用。总的来说，结果将阐明如何调节基本的DNA复制反应以保护基因组稳定并参与DNA修复。人类细胞已经发展出一种非常精确地复制DNA遗传信息的方法。它们使用专门设计的蛋白质来防止DNA中错误的形成，并在错误发生后纠正错误。这可以延长我们的寿命，减缓癌症的发生。了解DNA的复制和修复可以为我们提供进一步延缓衰老和癌症的方法，并开发创新的癌症治疗方法。负责复制我们DNA的蛋白质具有保护DNA编码信息的内在特性。我们正在努力了解它们用来保持我们基因组完整性和延缓衰老相关疾病和癌症发病的机制。我们的研究结果将阐明这一过程，并有助于确定可能成为减缓基因组退化药物目标的蛋白质功能。

DESCRIPTION (provided by applicant): The eukaryotic genome is synthesized by replication forks in which one strand is made as short segments, called Okazaki fragments, which are then joined. Each segment is initiated with an RNA primer, and then DNA is added to about 100 nucleotides. Polymerization from an upstream segment displaces the initiator RNA on the downstream segment into a flap. Flap endonuclease (FEN1) removes the flap, leaving a nick for ligation. Some flaps are thought to require additional cleavage by the Dna2 nuclease/helicase. Recent evidence also suggests that the Pif1 and Bloom (BLM) helicases have a role. Using genetic information as a guide, we have developed a biochemical system that reconstitutes Okazaki fragment joining. Early results show that while some flaps are readily processed by FEN1, others become long and require the action of Dna2. We will test whether Dna2 promotes correct processing of long flaps. Pif1 mutations suppress Dna2 mutations, suggesting that Pif1 creates a need for Dna2. BLM over expression suppresses Dna2 mutations, suggesting that BLM removes a need for Dna2. The functional relationships of these proteins will be determined. DNA added just after the RNA is thought to have frequent mismatch errors. We will determine whether strand displacement synthesis proceeds further into mismatch containing fragments, and is aided by Pif1, to allow 5' proofreading. Both FEN1 and Dna2 track onto the 5' ends of flaps and move to their points of cleavage, yet neither interferes with the action of the other. We will analyze motions of these two proteins to determine how this coordination is possible. BLM is a strong stimulator of DNA ligase I. The physical and functional interactions of the two proteins will be examined. Long patch base excision repair (LP BER) strongly resembles Okazaki fragment processing and also uses FEN1 and DNA ligase I. The 911 checkpoint complex, AP endonuclease and PCNA all coordinate and stimulate LP BER proteins. We will employ immunoprecipitation, substrate binding and activity stimulation assays to distinguish the roles of these three proteins in facilitation of the LP BER pathway. We will expand on preliminary results suggesting that DNA damage induces more effective interactions between 911 and LP BER proteins. Overall, results will clarify how fundamental DNA replication reactions are regulated to protect genome stability and participate in DNA repair. Human cells have developed a means of replicating the genetic information in their DNA in a very accurate manner. They use proteins that are specifically designed to prevent errors from forming in DNA and to correct any errors after they occur. This allows us to have a long lifespan and slows the onset of cancers. Understanding DNA replication and repair could provide us means to further delay aging and cancers, and to develop innovative cancer therapy. The proteins that are responsible for replicating our DNA have built-in properties that protect the information encoded in the DNA. We are working to understand the mechanisms that they employ to maintain the integrity of our genomes and delay the onset of aging-related diseases and cancers. Our results will clarify this process and also help identify protein functions that could be targets of agents designed to slow genome deterioration.