Kilbride E, Boocock M R, Stark W M
Institute of Biomedical and Life Sciences, University of Glasgow, 56 Dumbarton Road, Glasgow, G11 6NU, Scotland.
J Mol Biol. 1999 Jun 25;289(5):1219-30. doi: 10.1006/jmbi.1999.2864.
In order to investigate the functions of the parts of the Tn 3 recombination site res, we created hybrid recombination sites by placing the loxP site for Cre recombinase adjacent to the "accessory" resolvase-binding sites II and III of res. The efficiency and product topology of in vitro recombination by Cre between two of these hybrid sites were affected by the addition of Tn 3 resolvase. The effects of resolvase addition were dependent on the relative orientation and spacing of the elements of the hybrid sites. Substrates with sites II and III of res close to loxP gave specific catenated or knotted products (four-noded catenane, three-noded knot) when resolvase and Cre were added together. The product topological complexity increased when the length of the spacer DNA segment between loxP and res site II was increased. Similar resolvase-induced effects on Cre recombination product topology were observed in reactions of substrates with loxP sites adjacent to full res sites. The results demonstrate that the res accessory sites are sufficient to impose topological selectivity on recombination, and imply that intertwining of two sets of accessory sites defines the simple catenane product topology in normal resolvase-mediated recombination. They are also consistent with current models for the mechanism of catalysis by Cre.
为了研究Tn 3重组位点res各部分的功能，我们通过将Cre重组酶的loxP位点置于res的“辅助”解离酶结合位点II和III附近，构建了杂交重组位点。这些杂交位点中的两个之间由Cre介导的体外重组的效率和产物拓扑结构受到Tn 3解离酶添加的影响。解离酶添加的效果取决于杂交位点元件的相对取向和间距。当res的位点II和III靠近loxP时，在同时添加解离酶和Cre的情况下，底物会产生特定的连环或打结产物（四节点连环体、三节点结）。当loxP和res位点II之间的间隔DNA片段长度增加时，产物拓扑复杂性增加。在与完整res位点相邻的loxP位点的底物反应中，观察到了解离酶对Cre重组产物拓扑结构的类似影响。结果表明，res辅助位点足以对重组施加拓扑选择性，并暗示两组辅助位点的缠绕定义了正常解离酶介导的重组中简单连环体产物的拓扑结构。它们也与目前关于Cre催化机制的模型一致。
