Extreme C-terminal element of SprA serine integrase is a potential component of the “molecular toggle switch” which controls the recombination and its directionality,Molecular Microbiology

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Extreme C-terminal element of SprA serine integrase is a potential component of the “molecular toggle switch” which controls the recombination and its directionality
Molecular Microbiology ( IF 2.6 ) Pub Date : 2020-11-27 , DOI: 10.1111/mmi.14654
Kimihiro Abe ₁ , Takumi Takahashi ₂ , Tsutomu Sato _{1,

2}

Affiliation

In Bacillus subtilis, a sporulation-related gene, spsM, is disrupted by SPβ prophage, but reconstituted during sporulation through SPβ excision. The spsM reconstitution is catalyzed by a site-specific DNA recombinase, SprA, and its cognate recombination directionality factor, SprB. SprB interacts with SprA, directing the SprA-mediated recombination reaction from integration to excision; however, the details of the directionality control remains unclear. Here, we demonstrate the importance of the extreme C-terminal region (ECT) of SprA in the DNA recombination and directionality control. We created a series of SprA C-terminal deletants and examined their DNA-binding and recombination activities. Deletions in the ECT caused a loss of integration and excision activity, the magnitudes of which positively correlated with the deletion size. Gel shift study revealed that the loss of the integration activity was attributable to the failure of synaptic complex formation. The excision deficiency was caused by defective interaction with SprB. Moreover, alanine scanning analysis revealed that Phe532 is essential to interact with SprB. SprA_F532A, therefore, showed almost no excision activity, while retaining the integration activity. Collectively, these results suggest that the ECT plays the crucial roles in the interaction of SprA with SprB and possibly in the directional control of the recombination.

中文翻译：

SprA 丝氨酸整合酶的极端 C 端元件是控制重组及其方向性的“分子拨动开关”的潜在成分

在枯草芽孢杆菌中，孢子形成相关基因spsM被 SPβ 原噬菌体破坏，但在孢子形成过程中通过 SPβ 切除重建。该SPSM重组由位点特异性 DNA 重组酶 SprA 及其同源重组方向因子 SprB 催化。SprB 与 SprA 相互作用，指导 SprA 介导的重组反应从整合到切除；然而，方向性控制的细节仍不清楚。在这里，我们证明了 SprA 的极端 C 端区域 (ECT) 在 DNA 重组和方向性控制中的重要性。我们创建了一系列 SprA C 端缺失并检查了它们的 DNA 结合和重组活动。ECT 中的缺失导致整合和切除活动的丧失，其大小与缺失大小呈正相关。凝胶转移研究表明，整合活动的丧失是由于突触复合体形成失败。切除缺陷是由与 SprB 的相互作用缺陷引起的。此外，丙氨酸扫描分析表明 Phe532 是与 SprB 相互作用必不可少的。斯普林因此，_F532A几乎没有显示出切除活性，而保留了整合活性。总的来说，这些结果表明 ECT 在 SprA 与 SprB 的相互作用中以及可能在重组的方向控制中起着至关重要的作用。

更新日期：2020-11-27

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