The mechanism of DNA strand exchange by serine recombinases

丝氨酸重组酶进行DNA链交换的机制

• 批准号: BB/E022200/1
• 负责人: Marshall Stark
• 金额: $ 48.63万
• 依托单位: University of Glasgow
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE022200%2F1
• 关键词: mechanism; DNA; strand; exchange; serine

All living organisms contain immensely long double-helical DNA molecules that carry the information each cell needs to grow and multiply. This information is encoded in the sequence of the DNA building blocks called bases. Cellular machines translate the code into useful molecules such as proteins. Sometimes a cell changes the set of proteins that it makes in order to acquire new characteristics. For example, a disease-causing bacterium infecting a human being may switch the set of proteins displayed on its surface as a diguise to avoid detection and destruction by the human immune system. One way to do this is to rearrange (or 'edit') the DNA sequence by moving sections from place to place, or by adding/deleting sections. A special class of proteins called site-specific recombinases is dedicated to this 'cutting and pasting' role. To do it properly, the recombinase must identify the precise places in the DNA sequences that must be cut, then cut both DNA strands in each place and rejoin the cut ends in the required new arrangement. One family of site-specific recombinases has been proposed to do this reaction by a remarkable mechanism in which the DNA strands are first cut, then turned on a 'molecular wheel' to bring the cut ends into a new arrangement before rejoining them. This mechanism would be unprecedented in the world of enzymes, and has implications for the design and function of molecular machines, but it has not been proved. It is therefore very important to find out if this is really how the DNA is rearranged. In this project, we will use novel methods where we can see what is happening to the tiny individual recombinase 'machines' that are doing the reaction. We will attach pairs of fluorescent molecules to parts of the DNA in each machine, which we can see with a special type of microscope, and which will flash on and off as the bits of DNA move closer together or farther apart. We will also see what happens when we attach bits of the protein in the machine to the DNA to restrict their movement relative to each other. These experiments will allow us to find out the real mechanism of the reaction and analyse its properties, such as how fast it goes and what can interfere with it.

所有生物体都含有极长的双螺旋 DNA 分子，携带每个细胞生长和繁殖所需的信息。该信息被编码在称为碱基的 DNA 构建块的序列中。细胞机器将代码翻译成有用的分子，例如蛋白质。有时，细胞会改变其产生的一组蛋白质以获得新的特征。例如，感染人类的​​致病细菌可能会改变其表面展示的一组蛋白质作为伪装，以避免被人类免疫系统检测和破坏。实现此目的的一种方法是通过将部分从一个位置移动到另一个位置或通过添加/删除部分来重新排列（或“编辑”）DNA 序列。一类特殊的蛋白质，称为位点特异性重组酶，专门负责这种“剪切和粘贴”的作用。为了正确地做到这一点，重组酶必须识别 DNA 序列中必须切割的精确位置，然后切割每个位置的两条 DNA 链，并以所需的新排列重新连接切割端。一个位点特异性重组酶家族被提议通过一种非凡的机制来完成这种反应，其中DNA链首先被切割，然后打开“分子轮”，使切割端形成新的排列，然后重新连接。这种机制在酶的世界中是前所未有的，并且对分子机器的设计和功能具有影响，但尚未得到证实。因此，查明 DNA 是否确实如此重排非常重要。在这个项目中，我们将使用新颖的方法，我们可以看到正在进行反应的微小个​​体重组酶“机器”发生了什么。我们将把成对的荧光分子附着在每台机器中的 DNA 部分上，我们可以用一种特殊类型的显微镜看到它们，并且当 DNA 片段靠近或远离时，荧光分子会闪烁。我们还将看到当我们将机器中的蛋白质片段附着到 DNA 上以限制它们相对于彼此的运动时会发生什么。这些实验将使我们能够找出反应的真正机制并分析其特性，例如反应进行的速度以及什么会干扰它。

项目成果

Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination.

• DOI: 10.1093/nar/gkt1101
• 发表时间: 2014-02
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Colloms SD;Merrick CA;Olorunniji FJ;Stark WM;Smith MC;Osbourn A;Keasling JD;Rosser SJ
• 通讯作者: Rosser SJ

Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages.

• DOI: 10.1093/nar/gkv521
• 发表时间: 2015-07-13
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Olorunniji FJ;McPherson AL;Pavlou HJ;McIlwraith MJ;Brazier JA;Cosstick R;Stark WM
• 通讯作者: Stark WM

Intermediates in serine recombinase-mediated site-specific recombination.

丝氨酸重组酶介导的位点特异性重组的中间体。

• DOI: 10.1042/bst0390617
• 发表时间: 2011
• 期刊: Biochemical Society transactions
• 影响因子: 3.9
• 作者: Marshall Stark W

Architecture of a serine recombinase-DNA regulatory complex.

• DOI: 10.1016/j.molcel.2008.02.023
• 发表时间: 2008-04-25
• 期刊: MOLECULAR CELL
• 影响因子: 16
• 作者: Mouw, Kent W.;Rowland, Sally-J.;Gajjar, Mark M.;Boocock, Martin R.;Stark, W. Marshall;Rice, Phoebe A.
• 通讯作者: Rice, Phoebe A.

Synapsis and catalysis by activated Tn3 resolvase mutants.

• DOI: 10.1093/nar/gkn885
• 发表时间: 2008-12
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Olorunniji FJ;He J;Wenwieser SV;Boocock MR;Stark WM
• 通讯作者: Stark WM