Localization of Recombination sites in E. coli

描述（由申请人提供）：受损DNA和复制叉的修复对于基因组完整性和防止所有生物体生长期间的突变非常重要。E.大肠杆菌作为一个有价值的模型系统，了解如何重组修复发生在更复杂的真核细胞。更好地理解重组和DNA复制可以导致细菌感染和癌症的新疗法。复制叉停止在随机的，管家类型的DNA损伤。在大肠在大肠杆菌中，有证据表明RecA与停止的分叉结合并修复损伤，而不会诱导对DNA损伤的整体反应：SOS反应。当细胞暴露于外部DNA损伤时，RecA也会与这些分叉结合并修复它们。但在这种情况下，SOS被诱导。考虑到RecA在停止的分叉处与ssDNA的结合对于修复和诱导SOS响应都是至关重要的，该提议解决了为什么RecA在一种情况下诱导SOS，而不是另一种情况。有人提出，有分子机制，使细胞区分管家类型的DNA损伤和外部DNA损伤。这一点很重要，因为SOS反应是对危险环境的极端反应;不应该在标准生长情况下诱导。虽然在这两种情况下，DNA损伤的实际类型可能是相同的，但关键的区别在于细胞如何感知DNA损伤。进一步假设RecA和单链DNA结合蛋白（SSB）之间的相互作用在这一决定中至关重要。已经建立了三种试剂，揭示了RecA，SSB的位置和数量以及单个细胞中SOS表达的水平。它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：将构建RecA-YFP、SSB-CFP和sulAp-rfp（分别为黄色、青色和红色荧光蛋白），以便可以在单个细胞中同时测量所有三种。这对测试上述模型至关重要。这一建议的更广泛的影响是，人们将了解重组和DNA复制如何在细胞中发生时间和空间。这种理解对于开发细菌感染和癌症的新预防和治疗方法非常重要。这些新试剂也将为研究界的其他人提供重要的分子工具。

DESCRIPTION (provided by applicant): 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.