In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family
In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family
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DOI：
10.1073/pnas.95.10.5505
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发表时间：
1998-05-12
影响因子：
11.1
通讯作者：
Smith, MCM
中科院分区：
文献类型：
--
作者：
Thorpe, HM;Smith, MCM
已知宽宿主范围的温带链霉菌噬菌体phi C31的基因组通过作为位点特异性重组酶的解离酶/转化酶家族的成员的酶整合到宿主染色体中。在异源宿主大肠杆菌中，并在体外试验中，通过使用纯化的整合酶的重组特性，这种新型的整合酶上的噬菌体和生烃链霉菌附着位点，attP和attB，分别进行了研究。attP/B重组的产物，即，attL和attR与在S.产两性的。在体外测定中，仅需要缓冲液、纯化的整合酶和编码attP和attB的DNA，无论底物是超共沉淀的还是线性的，都会发生纯化。含有S12 F突变的突变整合酶在E. coli和体外培养。在attB/attB、attP/attP、attL/R或attB或attP与attL或attR的任何组合之间未观察到重组，表明原噬菌体的切除（attL/R重组）需要另外的噬菌体或链霉菌编码的因子。结果表明，在phi C31整合酶中，方向性受不同重组位点的严格控制，不需要形成解析酶/转化酶的典型拓扑结构。
The genome of the broad host range Streptomyces temperate phage, phi C31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase/invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and Streptomyces ambofaciens attachment sites, attP and attB, respectively, were investigated in the heterologous host, Escherichia coli, and in an in vitro assay by using purified integrase. The products of attP/B recombination, i.e., attL and attR, were identical to those obtained after integration of the prophage in S. ambofaciens. In the in vitro assay only buffer, purified integrase, and DNAs encoding attP and attB were required, Recombination occurred irrespective of whether the substrates were supercolied or linear. A mutant integrase containing an S12F mutation was completely defective in recombination both in E. coli and in vitro. No recombination was observed between attB/attB, attP/attP, attL/R, or any combination of attB or attP with attL or attR, suggesting that excision of the prophage (attL/R recombination) requires an additional phage- or Streptomyces-encoded factor. Recombination could occur intramolecularly to cause deletion between appropriately orientated attP and attB sites, The results show that directionality in phi C31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases/invertases.