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大型丝氨酸整合酶中方向性控制的结构基础

Structural basis of directionality control in large serine integrases.

作者信息

Shin Heewhan, Pigli Ying, Reyes Tania Peña, Fuller James R, Olorunniji Femi J, Rice Phoebe A

机构信息

Department of Biochemistry & Molecular Biology, The University of Chicago; Chicago IL, 60637, USA.

School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University; Liverpool, L3 3AF, UK.

出版信息

bioRxiv. 2025 Jan 13:2025.01.03.631226. doi: 10.1101/2025.01.03.631226.

DOI:10.1101/2025.01.03.631226
PMID:39803483
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11722253/
Abstract

Large serine integrases (LSIs) catalyze unidirectional site-specific DNA recombination reactions, yet those reactions are reversed by the presence of a cognate recombination directionality factor (RDF). Mechanistic understanding of directionality control has been hampered by a lack of structural information. Here, we use cryo-electron microscopy (cryo-EM) to determine the structures of six SPbeta integrase-DNA complexes along the integrative (-RDF) and excisive (+RDF) reaction pathways, at 4.16-7.18Å resolution. Our findings reveal how RDF-mediated repositioning of an integrase subdomain (1) dictates which pairs of DNA sites can be assembled into a synaptic complex to initiate recombination and (2) dictates which product complexes will be conformationally locked, preventing the back reaction. These mechanistic insights provide a conceptual framework for engineering efficient and versatile genome editing tools.

摘要

大型丝氨酸整合酶（LSIs）催化单向位点特异性DNA重组反应，但这些反应会因同源重组方向性因子（RDF）的存在而逆转。由于缺乏结构信息，对方向性控制的机制理解受到了阻碍。在这里，我们使用冷冻电子显微镜（cryo-EM），以4.16-7.18Å的分辨率确定了沿整合（-RDF）和切除（+RDF）反应途径的六个SPbeta整合酶-DNA复合物的结构。我们的研究结果揭示了RDF介导的整合酶亚结构域（1）的重新定位如何决定哪些DNA位点对可以组装成突触复合物以启动重组，以及（2）决定哪些产物复合物将在构象上被锁定，从而阻止逆向反应。这些机制上的见解为设计高效且通用的基因组编辑工具提供了一个概念框架。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/feb2692c8230/nihpp-2025.01.03.631226v2-f0007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/ba5df58503ed/nihpp-2025.01.03.631226v2-f0001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/b4f5fdd2bc2b/nihpp-2025.01.03.631226v2-f0002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/0d1a886d3a70/nihpp-2025.01.03.631226v2-f0003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/8146cd08ccad/nihpp-2025.01.03.631226v2-f0004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/a793d8173f2c/nihpp-2025.01.03.631226v2-f0005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/9efb8a649dcf/nihpp-2025.01.03.631226v2-f0006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/feb2692c8230/nihpp-2025.01.03.631226v2-f0007.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/ba5df58503ed/nihpp-2025.01.03.631226v2-f0001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/b4f5fdd2bc2b/nihpp-2025.01.03.631226v2-f0002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/0d1a886d3a70/nihpp-2025.01.03.631226v2-f0003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/8146cd08ccad/nihpp-2025.01.03.631226v2-f0004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/a793d8173f2c/nihpp-2025.01.03.631226v2-f0005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/9efb8a649dcf/nihpp-2025.01.03.631226v2-f0006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/05f7/11745111/feb2692c8230/nihpp-2025.01.03.631226v2-f0007.jpg

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本文引用的文献

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Bacteriophage lambda site-specific recombination.噬菌体 lambda 位点特异性重组。
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