MacDonald Alasdair I, Baksh Aron, Holland Alex, Shin Heewhan, Rice Phoebe A, Stark W Marshall, Olorunniji Femi J
School of Molecular Biosciences, University of Glasgow, Bower Building, Glasgow G12 8QQ, U.K.
School of Pharmacy and Biomolecular Sciences, Faculty of Science, Liverpool John Moores University, James Parsons Building, Byrom Street, Liverpool L3 3AF, U.K.
bioRxiv. 2024 Apr 3:2024.04.03.587898. doi: 10.1101/2024.04.03.587898.
Large serine integrases are phage- (or mobile element-) encoded enzymes that catalyse site-specific recombination reactions between a short DNA sequence on the phage genome () and a corresponding host genome sequence (), thereby integrating the phage DNA into the host genome. Each integrase has its unique pair of and sites, a feature that allows them to be used as orthogonal tools for genome modification applications. In the presence of a second protein, the Recombination Directionality Factor (RDF), integrase catalyses the reverse, excisive reaction, generating new recombination sites, and . In addition to promoting x reaction, the RDF inhibits x recombination. This feature makes the directionality of integrase reactions programmable, allowing them to be useful for building synthetic biology devices. In this report, we describe the degree of orthogonality of both integrative and excisive reactions for three related integrases (ϕC31, ϕBT1, and TG1) and their RDFs. Among these, TG1 integrase is the most active, showing near complete recombination in both x and x reactions, and the most directional in the presence of its RDF. Our findings show that there is varying orthogonality among these three integrases - RDF pairs: ϕC31 integrase was the least selective, with all three RDFs activating it for x recombination. Similarly, ϕC31 RDF was the least effective among the three RDFs in promoting the excisive activities of the integrases, including its cognate ϕC31 integrase. ϕBT1 and TG1 RDFs were noticeably more effective than ϕC31 RDF at inhibiting x recombination by their respective integrases, making them more suitable for building reversible genetic switches. AlphaFold-Multimer predicts very similar structural interactions between each cognate integrase - RDF pair. The binding surface on RDF is much more conserved than the binding surface on integrase, an indication that specificity is determined more by the integrase than the RDF. Overall, the observed weak integrase/RDF orthogonality across the three enzymes emphasizes the need for identifying and characterizing more integrase - RDF pairs. Additionally, the ability of a particular integrase's preferred reaction direction to be controlled to varying degrees by non-cognate RDFs provides a path to tunable, non-binary genetic switches.
大型丝氨酸整合酶是噬菌体（或移动元件）编码的酶，可催化噬菌体基因组上的短DNA序列（）与相应宿主基因组序列（）之间的位点特异性重组反应，从而将噬菌体DNA整合到宿主基因组中。每种整合酶都有其独特的和位点对，这一特性使其能够用作基因组修饰应用的正交工具。在第二种蛋白质重组方向性因子（RDF）存在的情况下，整合酶催化反向的切除反应，产生新的重组位点和。除了促进x反应外，RDF还抑制x重组。这一特性使整合酶反应的方向性具有可编程性，使其可用于构建合成生物学装置。在本报告中，我们描述了三种相关整合酶（ϕC31、ϕBT1和TG1）及其RDF的整合和切除反应的正交程度。其中，TG1整合酶活性最高，在x和x反应中均显示出近乎完全的重组，并且在其RDF存在下方向性最强。我们的研究结果表明，这三种整合酶-RDF对之间存在不同程度的正交性：ϕC31整合酶选择性最低，所有三种RDF都能激活它进行x重组。同样，ϕC31 RDF在促进整合酶的切除活性方面是三种RDF中效果最差的，包括其同源的ϕC31整合酶。ϕBT1和TG1 RDF在抑制各自整合酶的x重组方面明显比ϕC31 RDF更有效，使其更适合构建可逆遗传开关。AlphaFold-Multimer预测每个同源整合酶-RDF对之间的结构相互作用非常相似。RDF上的结合表面比整合酶上的结合表面保守得多，这表明特异性更多地由整合酶而非RDF决定。总体而言，观察到的这三种酶之间较弱的整合酶/RDF正交性强调了识别和表征更多整合酶-RDF对的必要性。此外，特定整合酶的首选反应方向能够被非同源RDF不同程度地控制，这为可调谐、非二元遗传开关提供了一条途径。
