Localization of Recombination sites in E. coli

描述(由申请人提供)：对受损的DNA和复制叉进行重组修复对于基因组完整性和防止所有生物生长过程中的突变非常重要。对于理解重组修复是如何在更复杂的真核细胞中发生的，大肠杆菌是一个有价值的模型系统。更好地了解重组和DNA复制可以为细菌感染和癌症带来新的治疗方法。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损伤的全球反应：SOS反应。当细胞暴露在外部DNA损伤时，RecA也会与这些叉子结合并修复它们。然而，在这种情况下，SOS被诱导。鉴于RecA在停止的叉子上与ssDNA的结合对于SOS反应的修复和诱导都是至关重要的，这一建议解决了为什么RecA在一种情况下诱导SOS，而在另一种情况下不诱导SOS。有人提出，有分子机制允许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；不应在标准增长情况下诱导这种反应。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。它进一步假设，RecA和单链DNA结合蛋白(SSB)之间的相互作用在这一决定中是关键的。已经建立了三种试剂来揭示RecA、SSB的位置和数量以及SOS在单个细胞中的表达水平。他们使用绿色荧光蛋白(GFP)。这些试剂包括RecA-GFP、SSB-GFP和SulAp-GFP。类似的试剂：RecA-YFP、SSB-CFP和SulAp-RFP(分别是黄色、青色和红色荧光蛋白)将被建立，这样人们就可以在单个细胞中同时检测这三种蛋白。这将是测试上述模型的关键。这一提议的更广泛的影响是，人们将学习重组和DNA复制如何在细胞中在时间和空间上发生。这一认识对于开发细菌感染和癌症的新预防和治疗方法将是重要的。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.