Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA.
Biochemistry. 2010 Apr 6;49(13):2786-95. doi: 10.1021/bi902204v.
The type I DNA topoisomerase from vaccinia virus (vTopo) forms a reversible covalent 3'-phosphotyrosyl linkage with a single strand of duplex DNA at the preferred sequence 5'-(C/T)CCTTp downward arrowN(-1)N(-2)N(-3)-3'. The enzyme-DNA covalent adduct is recombinogenic in cells, because the nicked strand downstream of the cleavage site can dissociate and be replaced by another DNA strand, potentially resulting in genome rearrangements if the enzyme executes strand ligation. Topo I could play an active role in strand exchange, either by altering the kinetics or thermodynamics of DNA strand binding or by serving as a proofreading gate to prevent ligation of incoming DNA strands containing mismatches. To address these questions, we have measured the kinetic and thermodynamic parameters for strand annealing to a purified vaccinia Topo I-DNA (vTopo-DNA) covalent complex containing a single-strand overhang and then compared them with the same overhang duplex in the absence of vTopo. We found that vTopo accelerates the strand association rate by 2-fold but has no effect on the rate of strand dissociation. vTopo has a similar small effect on the annealing parameters of a series of DNA strands containing single mismatches. In contrast, single base mismatches at the -1, -2, or -3 positions decreased the forward rate and equilibrium constant for reversible strand ligation by 10-fold. These data establish that while vTopo is a bystander during the annealing step of strand exchange, the enzyme strongly discriminates against mismatches close to the cleavage site during the subsequent events leading to strand ligation. A mechanism emerges where vTopo oscillates between an open state where the downstream DNA segment does not interact with the enzyme and a closed state where catalytically important contacts are formed with this region. This oscillation between an open and closed state of the covalently bound enzyme is likely important for regulating the number of DNA superhelical turns that are removed during the lifetime of the covalent complex with supercoiled substrates.
痘病毒 I 型 DNA 拓扑异构酶（vTopo）在其首选序列 5'-(C/T)CCTTp 箭头向下 N(-1)N(-2)N(-3)-3' 处与双链 DNA 的单链形成可逆的共价 3'-磷酸酪氨酸键。该酶-DNA 共价加合物在细胞中具有重组能力，因为切割部位下游的缺口链可以解离并被另一条 DNA 链取代，如果酶执行链连接，则可能导致基因组重排。拓扑异构酶 I 可以通过改变 DNA 链结合的动力学或热力学，或者作为一个防止带有错配的进入 DNA 链连接的校对门，在链交换中发挥积极作用。为了解决这些问题，我们已经测量了与包含单链突出的纯化痘病毒拓扑异构酶 I-DNA（vTopo-DNA）共价复合物退火的动力学和热力学参数，然后将其与没有 vTopo 的相同突出双链体进行了比较。我们发现 vTopo 将链缔合速率提高了 2 倍，但对链解离速率没有影响。vTopo 对包含单个错配的一系列 DNA 链的退火参数也有类似的微小影响。相比之下，-1、-2 或-3 位置的单个碱基错配使可逆链连接的正向速率和平衡常数降低了 10 倍。这些数据表明，虽然 vTopo 在链交换的退火步骤中是旁观者，但在导致链连接的后续事件中，酶强烈排斥靠近切割部位的错配。出现了一种机制，其中 vTopo 在开放状态和闭合状态之间振荡，在开放状态下，下游 DNA 片段不与酶相互作用，在闭合状态下，与该区域形成催化重要接触。这种共价结合酶的开放和闭合状态之间的振荡可能对于调节超螺旋底物上共价复合物存在期间去除的 DNA 超螺旋数很重要。
