The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site.

DNA连接酶（DNA ligases）在体内复制、修复与重组过程，以及体外分子生物学实验方案中均发挥不可或缺的重要作用。此前借助凝胶位移分析法（gel shift assays）对DNA连接酶开展的表征研究表明，切口位点的存在是酶与其双链DNA（dsDNA）底物实现紧密结合的必要条件，在不含切口的双链DNA上未检测到二者的相互作用。本研究中我们观察到显著的底物抑制效应，同时非切口型非底物双链DNA可抑制T4 DNA连接酶的自身腺苷酸化与切口封口两步反应。非底物DNA的抑制作用仅与总DNA浓度相关，而非DNA结构；当使用浓度为1 μg/mL的40聚体、75聚体或环状质粒DNA时，对连接反应的抑制效果完全一致。我们还发现，在高浓度非切口双链DNA环境中，T4 DNA连接酶的自身腺苷酸化速率降低了15倍以上。最后，我们通过电泳迁移率变动分析（EMSAs）证实，非底物型双链DNA可与带切口的双链DNA底物竞争酶结合位点。基于上述实验数据，我们提出假说：非切口双链DNA对T4 DNA连接酶的抑制作用，直接证明了两步切口结合机制的存在——即先发生一次不依赖切口的瞬时双链DNA结合事件，随后在切口位点存在时转变为稳定的结合复合物。