Mechanism of Termination of DNA Replication

DNA复制终止机制

• 批准号: 7989629
• 负责人: DEEPAK BASTIA
• 金额: $ 17.43万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-20 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7989629
• 关键词: Binding; Biochemical; Biochemistry; Crystallization; DNA Binding; DNA biosynthesis; Fission Yeast; Genetic Transcription; Goals; In Vitro; Investigation; Location; Modeling; Nature; Physiological; Protein Binding; Proteins; Recombinant DNA; Regulation; Request for Applications; Role; Saccharomyces cerevisiae; Saccharomycetales; Scheme; Site; Structure; Surgical Flaps; Testing; Work; Yeasts; gene cloning; genetic selection; helicase; in vivo; interest; mutant; protein complex; transcription factor; yeast genetics; yeast protein

We propose to continue our investigations into the mechanism of action of replication terminator proteins of the yeasts S. cerevisiae and the distantly related fission yeast Schizosaccharomyces pombe by using a combination of yeast genetics, in vivo and in vitro biochemistry and to start work towards the longer term goals of setting up crystallization trials of the terminator proteins bound to the terminus DMA. In S. cerevisiae we wish to uncover the mechanism of action of the Fob1 terminator protein by performing in vitro biochemical analysis to investigate if the protein is a contrahelicase that antagonizes the activities of eukaryotic replicative helicases. We also propose to work out the mechanism of action of the checkpoint proteins Tofl and Csm3 at the replication termini. We have discovered that the spacer region of rDNA of S. pombe has four not three replication arrest sites. Two of the four sites require the interesting protein Reb1 p that not only arrests forks in a polar mode but also terminates the transcription of rDNA and probably also acts as a transcription factor that turns on transcription. We wish to work out the mechanism of action of Reb1p. We have narrowed down the location of Ter1 of S. pombe to ~30bp and we wish to clone the gene that encodes the cognate terminator protein and investigate its mode of action. Finally, we wish to uncover the mode of action of the Swi1 and Swi3 proteins to determine if these act as "antisweepases".

我们建议继续研究复制终止蛋白的作用机制 酵母 S. cerevisiae 和远亲裂殖酵母 Schizosaccharomyces pombe 结合酵母遗传学、体内和体外生物化学并开始长期工作 建立与末端 DMA 结合的终止子蛋白的结晶试验的目标。在S。 我们希望通过体外实验来揭示Fob1终止子蛋白的作用机制 生化分析以研究该蛋白质是否是一种拮抗反解旋酶的活性 真核复制解旋酶。我们还建议制定检查点的作用机制 复制末端的 Tofl 和 Csm3 蛋白。我们发现 S. 粟酒有四个而不是三个复制停滞位点。四个位点中的两个需要有趣的蛋白质 Reb1 p 不仅以极性模式阻止分叉，而且终止 rDNA 的转录，并且可能还 作为启动转录的转录因子。我们希望研究出作用机制 Reb1p。我们已将粟酒裂殖酵母 Ter1 的位置缩小到约 30bp，我们希望克隆该基因 编码同源终止子蛋白并研究其作用模式。最后，我们希望揭开 Swi1 和 Swi3 蛋白的作用模式，以确定它们是否充当“抗扫描酶”。

项目成果

Helicase-contrahelicase interaction and the mechanism of termination of DNA replication.

解旋酶-反解旋酶相互作用和 DNA 复制终止机制。

• DOI: 10.1016/s0092-8674(00)81995-9
• 发表时间: 1996
• 期刊: Cell
• 影响因子: 64.5
• 作者: Manna,AC;Pai,KS;Bussiere,DE;Davies,C;White,SW;Bastia,D
• 通讯作者: Bastia,D

Investigations of pi initiator protein-mediated interaction between replication origins alpha and gamma of the plasmid R6K.

研究 pi 起始蛋白介导的质粒 R6K 复制起点 α 和 γ 之间的相互作用。

• DOI: 10.1074/jbc.m109.067439
• 发表时间: 2010
• 期刊: The Journal of biological chemistry
• 作者: Saxena,Mukesh;Singh,Samarendra;Zzaman,Shamsu;Bastia,Deepak
• 通讯作者: Bastia,Deepak

Termination of DNA replication in vitro: requirement for stereospecific interaction between two dimers of the replication terminator protein of Bacillus subtilis and with the terminator site to elicit polar contrahelicase and fork impedance.

体外 DNA 复制的终止：枯草芽孢杆菌复制终止子蛋白的两个二聚体之间以及与终止子位点之间立体特异性相互作用的要求，以引发极性反解旋酶和叉阻抗。

• DOI: 10.1002/j.1460-2075.1995.tb07038.x
• 发表时间: 1995
• 期刊: The EMBO journal
• 作者: Sahoo,T;Mohanty,BK;Patel,I;Bastia,D
• 通讯作者: Bastia,D

Replication initiation at a distance: determination of the cis- and trans-acting elements of replication origin alpha of plasmid R6K.

远距离复制起始：质粒 R6K 复制起点 α 的顺式和反式作用元件的测定。

• DOI: 10.1074/jbc.m109.067348
• 发表时间: 2010
• 期刊: The Journal of biological chemistry
• 作者: Saxena,Mukesh;Abhyankar,Mayuresh;Bastia,Deepak
• 通讯作者: Bastia,Deepak

Crystal structure of the replication terminator protein from B. subtilis at 2.6 A.

枯草芽孢杆菌复制终止子蛋白的晶体结构，2.6 A。

• DOI: 10.1016/0092-8674(95)90519-7
• 发表时间: 1995
• 期刊: Cell
• 影响因子: 64.5
• 作者: Bussiere,DE;Bastia,D;White,SW
• 通讯作者: White,SW