STUDIES ON THE MECHANISM OF GENETIC RECOMBINATION
基因重组机制的研究
基本信息
- 批准号:6289769
- 负责人:
- 金额:--
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:
项目摘要
The objective of this project is to uncover the molecular mechanisms of genetic rearrangements. The transposition reaction of bacteriophage Mu is studied as a model system. Critical steps in Mu transposition are a pair of DNA cleavages and strand transfers involving the ends of Mu DNA sequence and a target DNA; these reactions generate a branched DNA intermediate. The two chemical reaction steps take place within higher order protein-DNA complexes called transpososomes, the core of which is composed of two Mu-end DNA segments synapsed by a stably bound tetramer of MuA transposase protein. Transpososome assembly is controlled by a number of cofactors: an enhancer type DNA sequence element called IAS that overlaps the Mu operator sequence and the Mu repressor that binds to it, the MuB protein, the E. coli-encoded HU and IHF proteins, ATP, and Mg++. By making use of a simplified transpososome assembly reaction system, we have shown that both the Mu end DNA cleavage and the subsequent strand transfer at one Mu DNA end are catalyzed by the MuA monomer that is bound to the partner Mu DNA end within a transpososome. This in part explains why Mu DNA end synapsis is a prerequisite for the chemical steps. Roles of the IAS and MuB ATPase in the transpososome assembly process are currently investigated. For Tn10 and Mu transposition, two transposase monomers within the transpososome have been shown to catalyze all the chemical steps at the two transposon ends. By using chiral phosphorothioate containing DNA substrates, we compared the orientation of the substrate engagement at the transposase active site for the different reaction steps in both transposition reactions. MuB ATPase controls each of the early steps of Mu DNA transposition: it assists transpososome assembly, is involved in the target DNA site selection, activates the MuA transposase for strand transfer reaction, and protects transpososome from premature disassembly by ClpX chaperon protein until strand transfer is completed and the transposition intermediate is ready for DNA replication by the host replication proteins. In turn, the functional state of MuB is controlled by the ATPase cycle and by its interaction with MuA. Structural and functional aspects of MuB-DNA complex are currently under investigation by using a variety of physical and biochemical techniques. Efforts are continued toward solving the high-resolution structure of domains of MuA transposase as well as protein-DNA complexes in collaboration with scientists in LCP/NIDDK and at the University of Chicago. - genome rearrangements, DNA transposition, site-specific recombination, phage Mu.
该项目的目标是揭示基因重排的分子机制。以噬菌体Mu的转座反应为模型系统进行了研究。Mu转座的关键步骤是一对DNA切割和链转移,涉及Mu DNA序列的末端和靶DNA;这些反应产生分支的DNA中间体。这两个化学反应步骤发生在称为转座体的高级蛋白质-DNA复合物内,其核心由两个Mu末端DNA片段组成,所述DNA片段由稳定结合的MuA转座酶蛋白四聚体突触。转座体的装配受许多辅因子的控制:一种称为IAS的增强子型DNA序列元件,它与Mu操纵子序列和与之结合的Mu阻遏子重叠;大肠杆菌编码的HU和IHF蛋白、ATP和Mg++。通过利用一个简化的转座体组装反应系统,我们已经表明,无论是Mu末端DNA切割和随后的链转移在一个Mu DNA末端的催化的MuA单体结合到合作伙伴Mu DNA末端内的转座体内。这部分解释了为什么Mu DNA末端突触是化学步骤的先决条件。IAS和MuB ATP酶在转座体组装过程中的作用目前正在研究中。对于TnlO和Mu转座,转座体内的两个转座酶单体已显示催化两个转座子末端的所有化学步骤。通过使用含有手性硫代磷酸酯的DNA底物,我们比较了两种转座反应中不同反应步骤的转座酶活性位点处底物接合的方向。MuB ATP酶控制Mu DNA转座的每个早期步骤:它协助转座体组装,参与靶DNA位点选择,激活MuA转座酶进行链转移反应,并保护转座体免受ClpX伴侣蛋白过早分解,直到链转移完成,转座中间体准备好由宿主复制蛋白进行DNA复制。反过来,MuB的功能状态由ATP酶循环及其与MuA的相互作用控制。MuB-DNA复合物的结构和功能方面目前正在使用各种物理和生物化学技术进行研究。与LCP/NIDDK和芝加哥大学的科学家合作,继续努力解决MuA转座酶结构域以及蛋白质-DNA复合物的高分辨率结构。- 基因组重排,DNA转座,位点特异性重组,噬菌体Mu。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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KIYOSHI MIZUUCHI其他文献
KIYOSHI MIZUUCHI的其他文献
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{{ truncateString('KIYOSHI MIZUUCHI', 18)}}的其他基金
Study of the mechanism of bacterial chromosome partitioning systems
细菌染色体分配系统机制研究
- 批准号:
7967404 - 财政年份:
- 资助金额:
-- - 项目类别:
Study of the mechanism of septum localization during bacterial cell division
细菌细胞分裂过程中隔膜定位机制的研究
- 批准号:
8741432 - 财政年份:
- 资助金额:
-- - 项目类别:
Study of the mechanism of septum localization during bacterial cell division
细菌细胞分裂过程中隔膜定位机制的研究
- 批准号:
8349757 - 财政年份:
- 资助金额:
-- - 项目类别:
Study of the mechanism of bacterial chromosome partitioning systems
细菌染色体分配系统机制研究
- 批准号:
10250240 - 财政年份:
- 资助金额:
-- - 项目类别:
Study of the dynamics of higher order protein DNA complexes involved in variety of DNA transactions
研究参与各种 DNA 交易的高阶蛋白质 DNA 复合物的动力学
- 批准号:
10250238 - 财政年份:
- 资助金额:
-- - 项目类别:
Study of the DNA transposition target immunity at the single-molecule level
单分子水平DNA转座靶免疫研究
- 批准号:
7593577 - 财政年份:
- 资助金额:
-- - 项目类别:
Study of the mechanism of septum localization during bacterial cell division
细菌细胞分裂过程中隔膜定位机制的研究
- 批准号:
7593578 - 财政年份:
- 资助金额:
-- - 项目类别:
Study of the mechanism of septum localization during bacterial cell division
细菌细胞分裂过程中隔膜定位机制的研究
- 批准号:
7967402 - 财政年份:
- 资助金额:
-- - 项目类别:
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