Localization of Recombination sites in E. coli

受损DNA和复制分叉的重组修复对于基因组完整性和 防止所有有机体在生长过程中发生突变。大肠杆菌是一种有价值的模式系统 了解重组修复如何在更复杂的真核细胞中发生。更好 对重组和DNA复制的了解可以导致治疗细菌感染和 癌症。 复制分叉止步于随机的、内务类型的DNA损伤。在大肠杆菌中，有证据表明 RecA与停止的叉子结合并修复损伤，而不会引发对DNA的全球反应 损害：SOS反应。当细胞暴露于外部DNA损伤时，RecA也与这些结合 用叉子叉和修理它们。然而，在这种情况下，SOS被诱导。鉴于RecA与单链DNA在 停止的分叉对于修复和诱导SOS响应都至关重要，本提案解决了为什么RecA 在一种情况下诱导SOS，但在另一种情况下不诱导SOS。 有人提出，存在允许细胞区分家务管理的分子机制 DNA损伤的类型和外部DNA损伤。这很重要，因为SOS响应是一个 对危险环境的极端反应；不应被诱导为标准增长 情况。尽管在这两种情况下DNA损伤的实际类型可能是相同的，但关键是 不同之处在于细胞如何感知DNA损伤。进一步假设，这些相互作用 RecA和单链DNA结合蛋白(SSB)之间的关系在这一决定中至关重要。 建立了三种试剂，揭示了RecA、SSB的位置和数量以及SOS的水平 在单个细胞中表达。他们使用绿色荧光蛋白(GFP)。这些试剂包括 RecA-GFP、SSB-GFP和sulAp-GFP。类似试剂：RecA-YFP、SSB-CFP和SulAp-RFP(黄色、青色 和红色荧光蛋白)，以便一次可以同时测量所有这三种蛋白质 手机。这将是测试上述模型的关键。 这一提议的更广泛的影响是，人们将了解重组和DNA复制是如何发生的 在时间上和空间上在单元中。这一认识将对开发新的预防措施和 细菌感染和癌症的治疗。这些新试剂还将提供重要的分子 为研究社区中的其他人提供工具。

Recombinational repair of damaged DNA and replication forks is important for genomic integrity and the prevention of mutations during growth of all organisms. E. coli serves as a valuable model system for understanding how recombinational repair occurs in more complicated eucaryotic cells. Better understanding of recombination and DNA replication can lead to new treatments for bacterial infections and cancer. Replication forks stop at stochastic, housekeeping types of DNA damage. In E. coli, evidence suggests that RecA binds to stopped forks and repairs the damage without inducing the global response to DNA damage: the SOS Response. When cells are exposed to external DNA damage, RecA also binds to these forks and repairs them. However in this case, SOS is induced. Given that RecA's binding to ssDNA at a stopped fork is critical for both repair and induction of the SOS Response, this proposal addresses why RecA induces SOS in one situation, but not the other. It is proposed that there are molecular mechanisms that allow cells to discriminate between housekeeping types of DNA damage and external DNA damage. This is important because the SOS response is an extreme response to a hazardous environment; one that should not be induced for standard growth situations. Although the actual types of DNA damage can be identical under these two situations, the key difference is in how the cell perceives the DNA damage. It is further hypothesized that the interactions between RecA and the single-stranded DNA binding protein (SSB) are critical in this decision. Three reagents have been built that reveal the position and amount of RecA, SSB and levels of SOS expression in individual cells. They use the Green Fluorescent Protein (GFP). These reagents include RecA-GFP, SSB-GFP and sulAp-gfp. Similar reagents: RecA-YFP, SSB-CFP and sulAp-rfp (yellow, cyan and red fluorescent proteins respectively) will be built so that one can measure all three at once in a single cell. This will be critical to testing the above model. The broader impact of this proposal is that one will learn how recombination and DNA replication occur temporally and spatially in the cell. This understanding will be important for developing new preventions and treatments for bacterial infections and cancers. These new reagents will also provide important molecular tools for others in the research community.