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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(申请人提供)：FLP(‘Flip’)位点特异性重组酶由2微米圆形酵母质粒编码。重组的两个关键的化学前步骤是：(1)FLP识别靶DNA位点；(2)在FLP邻居之间建立功能性二聚体界面。通过定向进化，有可能放松或改变FLP的DNA特异性，并抑制削弱亚基间相互作用的突变。我们将确定“改变的特异性”和“抑制”的FLP变体的晶体结构与它们的同源DNA底物相关。2微米环在酵母中的持续存在是由一个分配系统和一个扩增系统介导的。FLP重组是后者的核心。我们将应用拓扑方法来确定FLP潜在的新活性，去连接(和解结)，这可能进一步有助于平等的质粒分割。FLP重组受制于重组伙伴(FRT位点)之间需要完美的间隔区同源性。正常情况下，这些部位在平面重组突触内呈反平行几何形状。一项证据表明，间隔区的异质性并不会取消重组；相反，它会促进甚至几轮的重组，以恢复间隔区的同源性和亲本构型。此外，建议FRT部位具有平行的几何形状。我们将设计拓扑测试来解决这一明显的矛盾。FLP的所有生化分析都是在裸DNA底物上进行的。然而，这个2微米的质粒是一种核驻留物质，被包装在染色质中。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。我们将完成几个正在进行中的机制研究，并启动新的研究，以阐明FLP在酵母体内的作用和调节。这些研究将促进我们对特定位点重组过程中的大分子识别和变构的理解。它们将为改进将特定部位重组作为基因工程工具的应用提供新的见解。最后，他们将在高阶染色质和天然2微米圆生理学的背景下为分析重组奠定基础。

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。