Alsaleh Abdulrazak, Holland Alexandria, Shin Heewhan, Reyes Tania Pena, Baksh Aron, Taiwo-Aiyerin Oluwateniola T, Pigli Ying, Rice Phoebe A, Olorunniji Femi J
School of Pharmacy & Biomolecular Sciences, Faculty of Health, Innovation, Technology, and Science, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, United Kingdom.
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, United States.
Nucleic Acids Res. 2025 Jan 24;53(3). doi: 10.1093/nar/gkaf050.
Recombination directionality factors (RDFs) for large serine integrases (LSIs) are cofactor proteins that control the directionality of recombination to favour excision over insertion. Although RDFs are predicted to bind their cognate LSIs in similar ways, there is no overall common structural theme across LSI RDFs, leading to the suggestion that some of them may be moonlighting proteins with other primary functions. To test this hypothesis, we searched for characterized proteins with structures similar to the predicted structures of known RDFs. Our search shows that the RDFs for two LSIs, TG1 integrase and Bxb1 integrase, show high similarities to a single-stranded DNA binding (SSB) protein and an editing exonuclease, respectively. We present experimental data to show that Bxb1 RDF is probably an exonuclease and TG1 RDF is a functional SSB protein. We used mutational analysis to validate the integrase-RDF interface predicted by AlphaFold2 multimer for TG1 integrase and its RDF, and establish that control of recombination directionality is mediated via protein-protein interaction at the junction of recombinase's second DNA binding domain and the base of the coiled-coil domain.
大型丝氨酸整合酶（LSIs）的重组方向性因子（RDFs）是辅助因子蛋白，可控制重组的方向性，使切除优于插入。尽管预计RDFs以相似的方式结合其同源LSIs，但LSI RDFs之间没有整体共同的结构主题，这表明它们中的一些可能是具有其他主要功能的兼职蛋白。为了验证这一假设，我们搜索了结构与已知RDFs预测结构相似的已表征蛋白。我们的搜索表明，两种LSIs（TG1整合酶和Bxb1整合酶）的RDFs分别与单链DNA结合（SSB）蛋白和编辑核酸外切酶高度相似。我们提供的实验数据表明，Bxb1 RDF可能是一种核酸外切酶，TG1 RDF是一种功能性SSB蛋白。我们使用突变分析来验证AlphaFold2多聚体预测的TG1整合酶及其RDF的整合酶-RDF界面，并确定重组方向性的控制是通过重组酶的第二个DNA结合结构域与卷曲螺旋结构域基部交界处的蛋白质-蛋白质相互作用介导的。
