Sequence of the loxP site determines the order of strand exchange by the Cre recombinase
Sequence of the loxP site determines the order of strand exchange by the Cre recombinase
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DOI：
10.1016/s0022-2836(02)01429-8
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发表时间：
2003-02-14
影响因子：
5.6
通讯作者：
Sadowski, PD
中科院分区：
文献类型：
--
作者：
Lee, L;Sadowski, PD
整合酶家族的保守位点特异性重组酶通过Holliday中间体进行重组。Cre重组酶是整合酶家族的一员，先前显示通过优先在其loxP靶序列的底链上切割和交换来启动重组。我们已经证实，使用野生型loxP位点和Cre蛋白的分子间重组反应存在这种链偏好。我们通过选择性地突变lox位点中的两个不对称易切割碱基对（那些紧邻Cre切割位点的碱基对）来研究这种链偏好的序列决定因素。我们发现，最初的链交换优先发生旁边的易切割的G残基。由此形成的霍利迪中间体的拆分优先发生在易分裂的A残基旁边。Lys 86，它接触的易切割的核苷酸在Cre-lox晶体结构，是重要的建立链偏好的决议的loxP-霍利迪中间体，但不是启动重组loxP网站之间。(C)2003爱思唯尔科技有限公司版权所有。
Conservative site-specific recombinases of the integrase family carry out recombination via a Holliday intermediate. The Cre recombinase, a member of the integrase family, was previously shown to initiate recombination by cleaving and exchanging preferentially on the bottom strand of its loxP target sequence. We have confirmed, this strand bias for an intermolecular recombination reaction that used wild-type loxP sites and Cre protein. We have examined the sequence determinants for this strand preference by selectively mutating the two asymmetric scissile base-pairs in the lox site (those immediately adjacent to the sites of cleavage by Cre). We found that the initial strand exchange occurs preferentially next to the scissile G residue. Resolution of the Holliday intermediate thus formed takes place preferentially next to the scissile A residue. Lys86, which contacts the scissile nucleotides in the Cre-lox crystal structures, was important for establishing the strand preference in the resolution of the loxP-Holliday intermediate, but not for the initiation of recombination between loxP sites. (C) 2003 Elsevier Science Ltd. All rights reserved.