MECHANISMS OF DNA REPLICATION

DNA 复制机制

基本信息

批准号：
7747960
负责人：
KENNETH J MARIANS
金额：
$ 67.09万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-07-01 至 2010-12-31
项目状态：
已结题

项目摘要

The repair of DNA damage is crucial to survival of all organisms. Thus, it is rfot surprising that the major DMA damage repair pathways, such as nucleotide excision repair and mismatch repair, are conserved from bacteria to man. These pathways are efficient and, for the most part, do not require the chromosomal DNA replication machinery for their activity. How do cells deal with the encounter between a replication fork and template DNA damage? What happens when the damage itself inactivates the replication fork?, creating a requirement for both replication fork restart and repair of the damage. As a result of studies from a number of groups, many centered, as ours have been, on the properties of PriA and its gene, a new paradigm has emerged describing the replication of the bacterial chromosome. This paradigm holds that even under normal growth conditions, the replication forks formed at oriC become inactivated as a result of an encounter with endogenous DNA template damage. This creates a requirement for both repair of the damage and reactivation of the replication forks. Our studies in the previous grant period have demonstrated that the <)>X174-type primosome is required for replication fork reactivation where it directs the assembly of a new replication fork on DNA substrates that are generated by the action of the recombination proteins. Furthermore, genetic data suggests that there are multiple pathways of replication fork restart involving different combinations of the primosomal proteins. In order to understand completely this intersection of two of the major pathways of DNA metabolism, we will model replication fork demise and reactivation in vitro. We will proceed by asking the following questions: What is the fate of the enzymatic components of the replication fork after a collision with either template DNA damage or a frozen protein-DNA complex? How is replication fork demise affected by the location and type of damage to the DNA? What are the DNA structures left at stalled replication forks? What conditions lead to DNA breakage at stalled replication forks? What is the biochemical basis for the existence of multiple pathways of replication fork restart? And, does the manner of recombination protein-directed processing of the DNA at a stalled fork direct the enzymatic pathway of replication forkreactivation? Using purified recombination and replication proteins, we will study the demise and reactivation of replication forks formed in isolated replisome complexes at oriC on small plasmid minichromosomes that have been engineered to carry specific types of DNA damage in specified locations.

DNA损伤的修复对于所有生物的存活至关重要。因此，主要DMA令人惊讶从细菌到人，损坏修复途径，例如核苷酸切除修复和不匹配修复。这些途径是有效的，并且在大多数情况下不需要染色体DNA复制机制活动。细胞如何处理复制叉和模板DNA损伤之间的相遇？什么时候会发生什么损坏本身使复制叉灭活，创造了复制叉重新启动和修复的要求损害。由于许多小组的研究，许多人像Pria及其特性一样集中了许多小组吉恩（Gene）出现了一种新的范式，描述了细菌染色体的复制。这个范式甚至在正常生长条件下，由于与内源性DNA模板损伤。这既需要修复损坏和重新激活复制叉。我们在上一个赠款期间的研究表明，需要<）> x174型原则。复制叉重新激活，它指导将新复制叉的组装在由由DNA底物上产生的DNA底物重组蛋白的作用。此外，遗传数据表明有多种复制途径叉子重启涉及原粒蛋白的不同组合。为了完全理解 DNA代谢的两种主要途径，我们将在体外建模复制叉灭活和重新激活。我们将通过询问以下问题来进行：复制的酶成分的命运是什么与模板DNA损伤或冷冻蛋白DNA复合物发生碰撞后的叉子？复制叉如何消亡受到DNA的位置和损害类型的影响？在停滞的复制叉处剩下的DNA结构是什么？什么条件会导致停滞的复制叉时DNA断裂？多重存在的生化基础是什么复制叉的途径重新启动？并且，DNA的重组蛋白定向加工的方式是否在A处停滞的叉子直接导致复制的酶促途径？使用纯化的重组和复制蛋白，我们将研究复制叉的灭亡和重新激活在孤立的重质体配合物上形成的小型质粒小质体，已设计为携带特定类型的DNA损伤在指定位置。