School of Molecular Biosciences, University of Glasgow, Bower Building, University Avenue, Glasgow, UK.
School of Chemistry, University of Glasgow, Joseph Black Building, University Avenue, Glasgow, UK.
Nat Commun. 2024 Nov 29;15(1):10407. doi: 10.1038/s41467-024-54531-4.
Serine recombinases are proposed to catalyse site-specific recombination by a unique mechanism called subunit rotation. Cutting and rejoining DNA occurs within an intermediate synaptic complex comprising a recombinase tetramer bound to two DNA sites. After double-strand cleavage at both sites, one half of the complex rotates 180° relative to the other, before re-ligation of the DNA ends. We used single-molecule FRET (smFRET) methods to provide compelling direct physical evidence for subunit rotation by recombinases Tn3 resolvase and Sin. Synaptic complexes containing fluorescently labelled DNA show FRET fluctuations consistent with the subunit rotation model. FRET changes were associated with the rotation steps, on a timescale of 0.4-1.1 , as well as opening and closing of the gap between the scissile phosphates during cleavage and ligation. Multiple rounds of recombination were observed within the ~25 s observation period, including frequent consecutive rotation events in the cleaved-DNA state without evidence of intermediate ligation.
丝氨酸重组酶被认为通过一种称为亚基旋转的独特机制来催化位点特异性重组。DNA 的切割和连接发生在包含与两个 DNA 位点结合的四聚体重组酶的中间突触复合物中。在两个位点的双链断裂后，复合物的一半相对于另一半旋转 180°，然后重新连接 DNA 末端。我们使用单分子 FRET（smFRET）方法为 Tn3 resolvase 和 Sin 重组酶的亚基旋转提供了令人信服的直接物理证据。含有荧光标记 DNA 的突触复合物显示出与亚基旋转模型一致的 FRET 波动。FRET 变化与旋转步骤相关，时间尺度为 0.4-1.1 ，以及在切割和连接过程中在剪切磷酸之间的间隙的打开和关闭。在大约 25 秒的观察时间内观察到多次重组循环，包括在没有中间连接证据的情况下在切割 DNA 状态下频繁连续的旋转事件。
