Zhang Likui
Marine Science & Technology Institute Department of Environmental Science and Engineering, Yangzhou University , No. 196 Huayang West Road, Hanjiang, Yangzhou, Jiangsu 225127, China.
Department of Biological Sciences, University of Alberta , Edmonton, Alberta T6G 2R3, Canada.
Chem Res Toxicol. 2017 Nov 20;30(11):1984-1992. doi: 10.1021/acs.chemrestox.7b00132. Epub 2017 Sep 15.
The bacteriophage T4 DNA polymerase (pol) and the closely related RB69 DNA pol have been developed into model enzymes to study family B DNA pols. While all family B DNA pols have similar structures and share conserved protein motifs, the molecular mechanism underlying natural drug resistance of nonherpes family B DNA pols and drug sensitivity of herpes DNA pols remains unknown. In the present study, we constructed T4 phages containing G466S, Y460F, G466S/Y460F, P469S, and V475W mutations in DNA pol. These amino acid substitutions replace the residues in drug-resistant T4 DNA pol with residues found in drug-sensitive herpes family DNA pols. We investigated whether the T4 phages expressing the engineered mutant DNA pols were sensitive to the antiviral drug phosphonoacetic acid (PAA) and characterized the in vivo replication fidelity of the phage DNA pols. We found that G466S substitution marginally increased PAA sensitivity, whereas Y460F substitution conferred resistance. The phage expressing a double mutant G466S/Y460F DNA pol was more PAA-sensitive. V475W T4 DNA pol was highly sensitive to PAA, as was the case with V478W RB69 DNA pol. However, DNA replication was severely compromised, which resulted in the selection of phages expressing more robust DNA pols that have strong ability to replicate DNA and contain additional amino acid substitutions that suppress PAA sensitivity. Reduced replication fidelity was observed in all mutant phages expressing PAA-sensitive DNA pols. These observations indicate that PAA sensitivity and fidelity are balanced in DNA pols that can replicate DNA in different environments.
噬菌体T4 DNA聚合酶（pol）以及与之密切相关的RB69 DNA聚合酶已被开发成为用于研究B族DNA聚合酶的模型酶。虽然所有B族DNA聚合酶都具有相似的结构并共享保守的蛋白质基序，但非疱疹B族DNA聚合酶天然耐药性和疱疹DNA聚合酶药物敏感性的分子机制仍不清楚。在本研究中，我们构建了在DNA聚合酶中含有G466S、Y460F、G466S/Y460F、P469S和V475W突变的T4噬菌体。这些氨基酸替换将耐药性T4 DNA聚合酶中的残基替换为在药物敏感的疱疹家族DNA聚合酶中发现的残基。我们研究了表达工程化突变DNA聚合酶的T4噬菌体是否对抗病毒药物膦甲酸（PAA）敏感，并对噬菌体DNA聚合酶的体内复制保真度进行了表征。我们发现G466S替换略微增加了PAA敏感性，而Y460F替换则赋予了耐药性。表达双突变G466S/Y460F DNA聚合酶的噬菌体对PAA更敏感。V475W T4 DNA聚合酶对PAA高度敏感，V478W RB69 DNA聚合酶也是如此。然而，DNA复制受到严重损害，这导致选择表达更强健的DNA聚合酶的噬菌体，这些聚合酶具有很强的DNA复制能力，并含有抑制PAA敏感性的额外氨基酸替换。在所有表达对PAA敏感的DNA聚合酶的突变噬菌体中都观察到复制保真度降低。这些观察结果表明，在能够在不同环境中复制DNA的DNA聚合酶中，PAA敏感性和保真度是平衡的。
