Integration and excision by the large serine recombinase φRv1 integrase
Integration and excision by the large serine recombinase φRv1 integrase
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DOI：
10.1111/j.1365-2958.2005.04517.x
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发表时间：
2005-03-01
影响因子：
3.6
通讯作者：
Hatfull, GF
中科院分区：
文献类型：
--
作者：
Bibb, LA;Hancox, MI;Hatfull, GF
结核分枝杆菌噬菌体样元件phiRv1利用丝氨酸重组酶家族的整合酶编码位点特异性重组系统。重组发生在假定的attP位点和宿主染色体之间，但不寻常的是，attB位点位于一个冗余重复元件（REP13E12）中，该元件在结核分枝杆菌基因组中有7个拷贝；其中四个元件含有适合phiRv1在体内整合的atb位点。虽然大丝氨酸整合酶的定向控制机制尚不清楚，但已经确定了phiRv1整合酶介导的体内特异性重组所需的重组方向性因子（RDF）。在这里，我们描述了phiRv1整合酶介导的整合和切除的体外重组反应，并表明phiRv1 RDF不仅是切除所必需的，而且抑制整合重组；这两种反应都不需要DNA超缠绕、宿主因子或高能辅助因子。整合、切除和切除介导的整合抑制需要简单的底物位点，这表明方向性的控制不涉及对酪氨酸整合酶所描述的高阶蛋白质- dna结构的操纵。
The Mycobacterium tuberculosis prophage-like element phiRv1 encodes a site-specific recombination system utilizing an integrase of the serine recombinase family. Recombination occurs between a putative attP site and the host chromosome, but is unusual in that the attB site lies within a redundant repetitive element (REP13E12) of which there are seven copies in the M. tuberculosis genome; four of these elements contain attB sites suitable for phiRv1 integration in vivo. Although the mechanism of directional control of large serine integrases is poorly understood, a recombination directionality factor (RDF) has been identified that is required for phiRv1 integrase-mediated excisive recombination in vivo. Here we describe defined in vitro recombination reactions for both phiRv1 integrase-mediated integration and excision and show that the phiRv1 RDF is not only required for excision but inhibits integrative recombination; neither reaction requires DNA supercoiling, host factors, or high-energy cofactors. Integration, excision and excise-mediated inhibition of integration require simple substrates sites, indicating that the control of directionality does not involve the manipulation of higher-order protein-DNA architectures as described for the tyrosine integrases.