Localization of Recombination sites in E. coli

大肠杆菌中重组位点的定位

• 批准号: 7595753
• 负责人: STEVEN J SANDLER
• 金额: $ 25.43万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-15 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7595753
• 关键词: Address; Affect; Area; Bacterial Infections; Binding; Biological Models; Cells; Chromosomes; Cloning; Communities; DNA; DNA Damage; DNA biosynthesis; DNA replication fork; Environment; Escherichia coli; Filament; Fluorescence Resonance Energy Transfer; Genes; Genetic Recombination; Genomics; Green Fluorescent Proteins; Growth; Housekeeping; In Vitro; Individual; Knowledge; Lead; Learning; Literature; Malignant Neoplasms; Measures; Methods; Modeling; Molecular; Monitor; Mutation; Organism; Phase; Positioning Attribute; Prevention; Principal Investigator; Proteins; Quantitative Microscopy; Reagent; Regulation; Research; SOS Response; SS DNA BP; Signal Transduction; Site; Son of Sevenless Proteins; Source; Testing; Time; Work; experience; fusion gene; in vivo; mutant; programs; recombinational repair; red fluorescent protein; repaired; response; tool; ultraviolet irradiation

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.

受损DNA和复制叉的重组修复对于基因组的完整性和基因的完整性至关重要