MECHANISMS OF SITE-SPECIFIC RECOMBINATION

位点特异性重组机制

基本信息

批准号：
7849881
负责人：
Makkuni JAYARAM
金额：
$ 15.57万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-18 至 2010-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The Flp ('flip') site-specific recombinase is coded for by the 2 micron circle yeast plasmid. Two key pre- chemical steps in recombination are: (1) recognition of the target DNA site by Flp and (2) establishing a functional dimer interface between Flp neighbors. By directed evolution, it is possible to relax or alter the DNA specificity of Flp and to suppress mutations that weaken intersubunit interactions. We will determine the crystal structures of 'altered specificity' and 'suppressor' Flp variants in association with their cognate DNA substrates. The persistence of the 2 micron circle in yeast is mediated by a partitioning system and an amplification system. Flp recombination is at the heart of the latter. We will apply topological methods to identify potential novel activities of Flp, decatenation (and unknotting), which may further assist in equal plasmid partitioning. Flp recombination is constrained by the need for perfect spacer homology between recombination partners (FRT sites). Normally, the sites assume antiparallel geometry within the planar recombination synapse. One piece of evidence suggests that spacer heterology does not abolish recombination; rather it promotes even rounds of recombination to restore spacer homology and parental configuration. Furthermore, the FRT sites are suggested to have a parallel geometry. We will devise topological tests to resolve this apparent contradiction. All biochemical analyses of Flp have been carried out with naked DNA substrates. The 2 micron plasmid, though, is a nuclear resident, packaged into chromatin. We will study features of recombination carried out in the context of nucleosome assembly and chromatin remodeling. We will complete several mechanistic studies in progress and initiate new studies to shed light on the action and regulation of Flp in vivo in yeast. These investigations will advance our understanding of macromolecular recognition and allostery during site-specific recombination. They will provide new insights for improved application of site-specific recombination as a tool for genetic engineering. Finally, they will set the stage for analyzing recombination in the context of high-order chromatin and native 2 micron circle physiology.

描述（由申请人提供）：FLP（'Flip'）位点特异性重组酶由2微米圆酵母质粒编码。重组的两个关键预化步骤是：（1）通过FLP识别目标DNA位点，（2）在FLP邻居之间建立功能二聚体界面。通过定向的进化，可以放松或改变FLP的DNA特异性并抑制弱化亚基间相互作用的突变。我们将确定与它们的同源DNA底物相关的“特异性变化”和“抑制剂” FLP变体的晶体结构。酵母中2微米圆的持久性是由分区系统和扩增系统介导的。 FLP重组是后者的核心。我们将采用拓扑方法来识别FLP，衰减（和无结）的潜在新活性，这可能会进一步有助于相等的质粒分配。 FLP重组受到重组伙伴（FRT站点）之间完美的间隔同源性的必要性的限制。通常，这些位点在平面重组突触中假定反平行的几何形状。一项证据表明，间隔者异质学不会废除重组。相反，它促进了甚至重组的圆形以恢复间隔同源性和父母的配置。此外，建议FRT位点具有平行的几何形状。我们将设计拓扑测试以解决这一明显的矛盾。所有FLP的生化分析均已使用裸DNA底物进行。但是，2微米质粒是包装成染色质的核居民。我们将研究在核小体组装和染色质重塑的背景下进行的重组的特征。我们将完成一些正在进行的机械研究，并启动新的研究，以阐明酵母中FLP体内FLP的作用和调节。这些研究将提高我们对特定地点重组期间大分子识别和变构的理解。他们将提供新的见解，以改善特定地点重组作为基因工程的工具。最后，他们将在高阶染色质和天然2微米圆形生理的背景下为分析重组奠定阶段。

项目成果

期刊论文数量（19）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Sequence-specific cleavage of DNA via nucleophilic attack of hydrogen peroxide, assisted by Flp recombinase.

在 Flp 重组酶的协助下，通过过氧化氢的亲核攻击对 DNA 进行序列特异性切割。

DOI：
10.1021/bi00069a002
发表时间：
1993
期刊：
Biochemistry
影响因子：
2.9
作者：
Kimball,AS;Lee,J;Jayaram,M;Tullius,TD
通讯作者：
Tullius,TD

Electrostatic suppression allows tyrosine site-specific recombination in the absence of a conserved catalytic arginine.

静电抑制允许在不存在保守的催化精氨酸的情况下进行酪氨酸位点特异性重组。

DOI：
10.1074/jbc.m110.112292
发表时间：
2010
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Rowley,PaulA;Kachroo,AashiqH;Ma,Chien-Hui;Maciaszek,AnnaD;Guga,Piotr;Jayaram,Makkuni
通讯作者：
Jayaram,Makkuni

Mutations that improve the binding of yeast FLP recombinase to its substrate.

提高酵母 FLP 重组酶与其底物结合的突变。