MECHANISMS OF DNA REPLICATION

DNA损伤的修复对所有生物体的生存至关重要。因此，令人惊讶的是，主要的DMA 损伤修复途径，如核苷酸切除修复和错配修复，从细菌到人类都是保守的。 这些途径是有效的，而且在大多数情况下，不需要染色体dna复制机制来实现其 活动。细胞如何应对复制分叉和模板DNA损伤之间的遭遇？当发生以下情况时会发生什么 损坏本身会停用复制分叉？，因此需要重新启动复制分叉并修复 损坏。作为一些研究小组研究的结果，许多研究小组像我们一样，集中在PriA及其 基因，一种描述细菌染色体复制的新范式已经出现。这一范式认为，即使 在正常生长条件下，在ORIC形成的复制分叉由于遇到 内源性DNA模板损伤。这就产生了修复损坏和重新激活 复制分叉。我们在前一个资助期的研究表明，)&gt；x174型原粒是 复制分叉重新激活，它指导在DNA底物上组装新的复制分叉，DNA底物由 重组蛋白的作用。此外，遗传数据表明，有多种复制途径 分叉重新启动涉及不同的原始体蛋白组合。为了完全理解这一交集 DNA代谢的两条主要途径，我们将在体外模拟复制叉的死亡和重新激活。 我们将继续提出以下问题：复制的酶成分的命运是什么 与模板DNA损伤或冻结的蛋白质-DNA复合体碰撞后的叉子？复制分叉是如何消亡的 受DNA损伤的位置和类型的影响？停滞不前的复制叉子上剩下的DNA结构是什么？什么 在停滞的复制叉子上，条件会导致DNA断裂吗？多重细菌存在的生物化学基础是什么？ 复制分叉的路径重新启动？而且，DNA的重组蛋白质导向的处理方式是否在一次 停滞不前的叉子直接复制叉子重新激活的酶路径？ 利用纯化的重组和复制蛋白，我们将研究复制叉子的死亡和重新激活 在小质粒微染色体上形成的在ORIC上分离的复制体复合体，这些微染色体被设计成携带 特定位置的特定类型的DNA损伤。

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.