Comparative analysis of the end-joining activity of several DNA ligases

DNA ligases catalyze the repair of phosphate backbone breaks in DNA, acting with highest activity on breaks in one strand of duplex DNA. Some DNA ligases have also been observed to ligate two DNA fragments with short complementary overhangs or blunt-ended termini. In this study, several wild-type DNA ligases (phage T3, T4, and T7 DNA ligases, Paramecium bursaria chlorella virus 1 (PBCV1) DNA ligase, human DNA ligase 3, and Escherichia coli DNA ligase) were tested for their ability to ligate DNA fragments with several difficult to ligate end structures (blunt-ended termini, 3′- and 5′- single base overhangs, and 5′-two base overhangs). This analysis revealed that T4 DNA ligase, the most common enzyme utilized for in vitro ligation, had its greatest activity on blunt- and 2-base overhangs, and poorest on 5′-single base overhangs. Other ligases had different substrate specificity: T3 DNA ligase ligated only blunt ends well; PBCV1 DNA ligase joined 3′-single base overhangs and 2-base overhangs effectively with little blunt or 5′- single base overhang activity; and human ligase 3 had highest activity on blunt ends and 5′-single base overhangs. There is no correlation of activity among ligases on blunt DNA ends with their activity on single base overhangs. In addition, DNA binding domains (Sso7d, hLig3 zinc finger, and T4 DNA ligase N-terminal domain) were fused to PBCV1 DNA ligase to explore whether modified binding to DNA would lead to greater activity on these difficult to ligate substrates. These engineered ligases showed both an increased binding affinity for DNA and increased activity, but did not alter the relative substrate preferences of PBCV1 DNA ligase, indicating active site structure plays a role in determining substrate preference.DNA连接酶（DNA ligase）可催化DNA磷酸骨架断裂的修复，对双链DNA单链断裂位点展现出最高的催化活性。部分DNA连接酶还被观测到可连接带有短互补突出端或平末端的两个DNA片段。本研究选取了多种野生型DNA连接酶，包括噬菌体T3、T4、T7 DNA连接酶，绿草履虫小球藻病毒1型（Paramecium bursaria chlorella virus 1, PBCV1）DNA连接酶，人DNA连接酶3以及大肠杆菌DNA连接酶，对其连接带有多种难连接末端结构的DNA片段的能力进行了测试，所测试的末端结构包括平末端、3′端与5′端单碱基突出端以及5′端双碱基突出端。分析结果显示，作为体外连接实验中最常用的酶，T4 DNA连接酶在平末端与双碱基突出端上活性最高，而在5′端单碱基突出端上活性最低。其他连接酶则展现出不同的底物特异性：T3 DNA连接酶仅对平末端具有较高的连接活性；PBCV1 DNA连接酶可有效连接3′端单碱基突出端与双碱基突出端，几乎无平末端与5′端单碱基突出端的连接活性；人DNA连接酶3则对平末端与5′端单碱基突出端活性最高。不同DNA连接酶在平末端DNA上的催化活性与其在单碱基突出端上的活性之间并无相关性。此外，研究人员将DNA结合结构域（Sso7d、人DNA连接酶3锌指结构域（hLig3 zinc finger）以及T4 DNA连接酶N端结构域（T4 DNA ligase N-terminal domain））与PBCV1 DNA连接酶进行融合，以探究增强DNA结合能力是否可提升其在这些难连接底物上的催化活性。这些经工程改造的连接酶不仅展现出对DNA更高的结合亲和力，催化活性也有所提升，但并未改变PBCV1 DNA连接酶原本的相对底物偏好，这表明活性位点结构在决定底物偏好性中发挥关键作用。