Enzymatic Mechanisms of DNA Synthesis

描述(由申请人提供)： 哺乳动物的染色体DNA是在复制叉子上合成的，在复制叉子中，一条链被制成短片段，称为冈崎片段，然后连接在一起。我们正在通过与纯化的哺乳动物蛋白重组，并使用使用类似复制系统的酿酒酵母的遗传工具来确定连接反应的机制。冈崎片段由一小段RNA起始，然后延长到大约100个核苷酸。根据目前的模型，从上游片段聚合而来的下游片段的RNA区域被置换成瓣。在结扎之前，该瓣被认为是被DNA2核酸酶/解旋酶(DNA2p)和瓣内切酶(FEN1)移除的。然而，一些证据表明，在没有DNA2p的情况下，皮瓣被移除。我们将使用重构来确定DNA2p是否提高了导致连接的反应的效率。我们还将进一步定义Dna 2p的机制，以确定其5‘翻盖进入、解旋酶跟踪和切割模式是否表明它与FEN1顺序工作。FEN1在瓣的底部裂开，形成结扎底物。我们发现它能识别襟翼的5‘端，然后移动到底部。在体内，该瓣可以通过分支迁移到各种中间体来平衡，但只有一种是FEN1底物。急冷流动力学分析将确定FEN1是等待正确的构型还是诱导其形成。FEN1有一个灵活的环结构，被认为参与了对切割位点的跟踪。对突变体的分析应该揭示这种结构的作用。一个提出的在体细胞中扩展重复序列的模型暗示了在去瓣过程中形成的中间底物结构。我们最近部分地重组了这种类型的扩张，得出的结论是，DNA连接酶I和FEN1催化的反应之间的竞争决定了扩张的可能性。为了验证我们的结论，我们将使用一种报告基因表达被扩张扰乱的质粒，在体内探索酿酒酵母的扩张机制。DNA连接酶I的细胞表达将发生变化，以确定连接和FEN1裂解之间竞争的影响。哺乳动物长斑块碱基切除修复使用与Okazaki片段处理相同的蛋白质。它还包括AP内切酶(APE1)，我们发现它是一种协调和刺激因子。APE1刺激FEN1和DNA连接酶I，并通过该途径促进底物的移动。结合和动力学分析将被用来确定APE1定向刺激的机制。

DESCRIPTION (provided by applicant): Mammalian chromosomal DNA is synthesized at replication forks in which one strand is made as short segments, called Okazaki fragments, that are later joined. We are defining the mechanism of the joining reaction by reconstitution with purified mammalian proteins, and using genetic tools from S. cerevisiae, which employs a similar replication system. Okazaki fragments are initiated by a short segment of RNA and then elongated to about 100 nucleotides. By current models, polymerization from an upstream fragment displaces the RNA region of the downstream fragment into a flap. The flap is thought to be removed by Dna2 nuclease/helicase (Dna2p) and flap endonuclease (FEN1), prior to ligation. However, some evidence suggests that the flap is removed without Dna2p. We will use reconstitution to determine whether Dna2p improves the efficiency of the reactions leading to joining. We will also further define the mechanism of Dna2p to establish whether its 5' flap entry, helicase tracking and cleavage pattern indicate that it works in sequence with FEN1. FEN1 cleaves at the base of the flap to create the ligation substrate. We showed that it recognizes the 5' end of the flap and then moves to the base. In vivo, the flap can equilibrate by branch migration to a variety of intermediates, but only one is a FEN1 substrate. Quench flow kinetic analyses will determine whether FEN1 waits for the correct configuration or induces its formation. FEN1 has a flexible loop structure thought to be involved in tracking to the cleavage site. Analysis of mutants should reveal the role of this structure. A proposed model for expansion of repeat sequences in somatic cells implicates intermediate substrate structures formed during the flap removal. We recently partially reconstituted this type of expansion, concluding that a competition between reactions catalyzed by DNA ligase I and FEN1 determines expansion probability. To verify our conclusions, expansion mechanisms will be probed in vivo in S. cerevisiae using a plasmid in which reporter gene expression is disrupted by expansion. Cellular expression of DNA ligase I will be varied to determine the effects of competition between ligation and FEN1 cleavage. Mammalian long patch base excision repair uses the same proteins as Okazaki fragment processing. It also includes AP endonuclease (APE1) which we found to be a coordinating and stimulatory factor. APE1 stimulates both FEN1 and DNA ligase I, and promotes movement of the substrate through the pathway. Binding and kinetic analyses will be employed to determine the mechanisms of APE1 directed stimulation.