Localization of Recombination sites in E. coli

大肠杆菌中重组位点的定位

• 批准号: 7796802
• 负责人: STEVEN J SANDLER
• 金额: $ 25.2万
• 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 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7796802
• 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和复制叉的修复对于基因组的完整性和基因的表达是重要的。 防止所有生物体在生长过程中发生突变。E.大肠杆菌作为一个有价值的模型系统， 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复制的理解可能会导致细菌感染的新疗法， 癌 复制叉停止在随机的，管家类型的DNA损伤。在大肠有证据表明， RecA与停止的分叉结合并修复损伤，而不诱导对DNA的整体反应， 紧急救援：SOS响应。当细胞暴露于外部DNA损伤时，RecA也与这些DNA结合。 叉和修理他们。但在这种情况下，SOS被诱导。考虑到RecA与ssDNA的结合速度 停止的分叉对于修复和诱导SOS响应都是至关重要的，这个建议解决了为什么RecA 在一种情况下会引发SOS，但在另一种情况下不会。 有人提出，有分子机制，使细胞区分管家 DNA损伤和外部DNA损伤。这一点很重要，因为SOS响应是一个 对危险环境的极端反应;不应该被诱导为标准生长的反应 situations.虽然在这两种情况下，DNA损伤的实际类型可能是相同的，但关键是 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，可以揭示RecA，SSB的位置和数量以及SOS的水平 在单个细胞中表达。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：RecA-YFP、SSB-CFP和sulAp-rfp（黄色、青色 和红色荧光蛋白）将被建造，以便人们可以在一个单一的测量所有三个一次 cell.这对测试上述模型至关重要。 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 在细胞中的时间和空间。这种理解对于开发新的预防措施和 治疗细菌感染和癌症。这些新试剂还将提供重要的分子生物学 为研究界的其他人提供工具。

项目成果

Differential requirements of two recA mutants for constitutive SOS expression in Escherichia coli K-12.

两种recA突变体在大肠杆菌K-12中对SOS组成型表达的不同要求。

• DOI: 10.1371/journal.pone.0004100
• 发表时间: 2008
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Long,JarukitEdward;Renzette,Nicholas;Centore,RichardC;Sandler,StevenJ
• 通讯作者: Sandler,StevenJ

RecA4142 causes SOS constitutive expression by loading onto reversed replication forks in Escherichia coli K-12.

RecA4142 通过加载到大肠杆菌 K-12 中的反向复制叉上，引起 SOS 组成型表达。

• DOI: 10.1128/jb.01623-09
• 发表时间: 2010
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Long,JarukitEdward;Massoni,ShawnC;Sandler,StevenJ
• 通讯作者: Sandler,StevenJ

Specificity in suppression of SOS expression by recA4162 and uvrD303.

recA4162 和 uvrD303 抑制 SOS 表达的特异性。

• DOI: 10.1016/j.dnarep.2013.09.003
• 发表时间: 2013
• 期刊: DNA repair
• 影响因子: 3.8
• 作者: Massoni,ShawnC;Sandler,StevenJ
• 通讯作者: Sandler,StevenJ

Suppression of constitutive SOS expression by recA4162 (I298V) and recA4164 (L126V) requires UvrD and RecX in Escherichia coli K-12.

在大肠杆菌 K-12 中，recA4162 (I298V) 和 recA4164 (L126V) 抑制 SOS 的组成型表达需要 UvrD 和 RecX。

• DOI: 10.1111/j.1365-2958.2009.06765.x
• 发表时间: 2009
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Long,JarukitE;Renzette,Nicholas;Sandler,StevenJ
• 通讯作者: Sandler,StevenJ

RecA-mediated SOS induction requires an extended filament conformation but no ATP hydrolysis.

• DOI: 10.1111/j.1365-2958.2008.06341.x
• 发表时间: 2008-09
• 期刊: MOLECULAR MICROBIOLOGY
• 影响因子: 3.6
• 作者: Gruenig, Marielle C.;Renzette, Nicholas;Long, Edward;Chitteni-Pattu, Sindhu;Inman, Ross B.;Cox, Michael M.;Sandler, Steven J.
• 通讯作者: Sandler, Steven J.