Dean F B, Cozzarelli N R
J Biol Chem. 1985 Apr 25;260(8):4984-94.
We studied the interaction between topoisomerase I and a nicked DNA substrate to determine how the nick permits Escherichia coli topoisomerase I to catenate and knot duplex DNA rings. The presence of just a single nick in a 6600-base pair DNA increased the amount of DNA bound to topoisomerase I by 6-fold. The enzyme acts at the nick, as shown by linearization of nicked circles and covalent attachment of an enzyme molecule opposite the nick. DNA breaks are also introduced by the enzyme at sites not opposite to a nick, but three orders of magnitude less efficiently. The break induced by the enzyme is within several base pairs of the nick and on the complementary strand, but the exact site cut is dictated by DNA sequence requirements. Because these sequence requirements are identical to those for cutting of single-stranded DNA, we conclude that the enzyme stabilizes a denatured region at the nick. Breaks in single-stranded DNA occur 98% of the time when a C residue is four bases to the 5' side unless G is adjacent and 5' to the break. For a DNA circle nicked at a unique location, the efficiency of DNA breakage opposite the nick correlates with the rate of catenation. We present a unified model for the relaxation, catenation, and knotting reactions of topoisomerase I in which the enzyme induces a break in a single-stranded region, but bridges that break with covalent and noncovalent interactions and allows passage of one duplex or single-stranded DNA segment.
我们研究了拓扑异构酶I与带切口的DNA底物之间的相互作用，以确定切口如何使大肠杆菌拓扑异构酶I将双链DNA环连环化并打结。在一个6600个碱基对的DNA中仅存在一个切口，就会使与拓扑异构酶I结合的DNA量增加6倍。如带切口的环状DNA的线性化以及酶分子在切口对面的共价连接所示，该酶作用于切口处。该酶也会在与切口不相对的位点引入DNA断裂，但效率要低三个数量级。酶诱导的断裂位于切口的几个碱基对范围内且在互补链上，但确切的切割位点由DNA序列要求决定。由于这些序列要求与切割单链DNA的序列要求相同，我们得出结论，该酶在切口处稳定了一个变性区域。当一个C残基在断裂位点5'侧四个碱基处时，单链DNA断裂发生的概率为98%，除非G与断裂位点相邻且在其5'侧。对于在唯一位置带切口的DNA环，切口对面的DNA断裂效率与连环化速率相关。我们提出了一个关于拓扑异构酶I的松弛、连环化和打结反应的统一模型，其中该酶在单链区域诱导一个断裂，但通过共价和非共价相互作用连接该断裂，并允许一个双链或单链DNA片段通过。
