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首页> 外文期刊>Journal of bacteriology >Piv Site-Specific Invertase Requires a DEDD Motif Analogous to the Catalytic Center of the RuvC Holliday Junction Resolvases
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Piv Site-Specific Invertase Requires a DEDD Motif Analogous to the Catalytic Center of the RuvC Holliday Junction Resolvases

机译：Piv特定位点的转化酶需要DEDD基序，类似于RuvC霍利迪交汇点催化的催化中心

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摘要

Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine- or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.
机译：Piv是一种独特的原核位点特异性DNA转化酶，与IS 110 / IS 492 家族的插入元件的转座酶有关，并且与该位点特异性没有相似性酪氨酸或丝氨酸重组酶家族的重组酶。预测的三级结构包括RNase H样折叠，该折叠通常包含逆转录病毒整合酶超家族的重组酶或核酸酶的催化​​位点，包括转座酶和RuvC样的Holliday连接分辨物。分别类似于转座酶和RuvC的DDE和DEDD催化基序，四个Piv酸性残基D9，E59，D101和D104似乎适当地位于RNase H折叠内，以配位两个二价金属阳离子。这表明Piv介导的位点特异性倒置与逆转录病毒整合酶超家族的酶催化的转座或内切核酸反应之间的机理相似性。 DEDD基序在Piv催化活性中的作用通过使用Piv变体解决，该变体被单独取代或在这些酸性残基处多重取代，并检测体内转化，分子间重组和DNA结合活性。结果表明，DEDD基序的所有四个残基都是Piv催化活性所必需的。 DEDD残基对于 inv 重组位点的识别和结合不是必需的，但是这种酸性四聚体确实有助于Piv- inv 相互作用的稳定性。基于这些结果，提出了包括霍利迪交界处分辨率在内的Piv介导反演的工作模型。

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