Cre mutants with altered DNA binding properties
Cre mutants with altered DNA binding properties
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DOI：
10.1074/jbc.273.36.22884
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发表时间：
1998-09-04
影响因子：
4.8
通讯作者：
Kisters-Woike, B
中科院分区：
文献类型：
--
作者：
Hartung, M;Kisters-Woike, B
噬菌体 P1 的重组酶 Ore 是位点特异性重组酶和整合酶家族的成员，可催化分子间和分子内 DNA 重排。为了了解该蛋白质如何特异性识别其靶序列，我们构建了在假定 DNA 结合区的不同位置进行氨基酸取代的 Ore 突变体。在这里，我们展示了这些 Cre 突变体的体外 DNA 结合和体内重组实验的结果。在体外测试中，大多数假定的 DNA 结合氨基酸的取代导致靶标结合丧失或靶标识别特异性扩大。在导致靶标特异性扩大的突变中，其中之一 N317A 会导致与野生型 loxP 靶标的重组效率降低，但与野生型 Ore 相比，在体内实验中，它会与野生型 LoxP 靶标重组。 野生型靶序列的对称变体。该目标变体与野生型 loxP 的不同之处在于反向重复序列第 6 位的对称 C 替换为 A。我们为整合酶和重组酶家族提出了一个常见的多螺旋 DNA 结合基序，该模型意味着 lambda 整合酶的 DNA 结合区域发生主要结构重排，类似于其他蛋白质在接触目标 DNA 时 DNA 结合基序的结构重排。
The recombinase Ore of bacteriophage P1 is a member of the family of site-specific recombinases and integrases that catalyze inter- and intramolecular DNA rearrangements, To understand how this protein specifically recognizes its target sequence, we constructed Ore mutants with amino acid substitutions in different positions of the presumptive DNA binding region. Here we present the results of in vitro DNA binding and in vivo recombination experiments with these Cre mutants. Most substitutions of presumptive DNA-binding amino acids in in vitro tests resulted either in the loss of target binding or in a broadening of target recognition specificity, Of the mutations resulting in a broadening of target specificity, one, N317A, results in a reduced recombination efficiency with the wild-type loxP target but recombines, in contrast to wild-type Ore, in in vivo experiments, with a symmetric variant of the wild-type target sequence. This target variant differs from wildtype loxP by the symmetric C to A replacement in position 6 of the inverted repeats. We propose a common multihelical DNA binding motif for the family of integrases and recombinases, This model implies a major structural rearrangement for the DNA binding region of lambda integrase, analogous to the structural rearrangements of the DNA binding motifs of other proteins when contacting their target DNA.