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.

中文翻译：

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磷酸乙酸敏感的T4 DNA聚合酶揭示了对DNA聚合酶功能的新见解。

噬菌体T4 DNA聚合酶（pol）和密切相关的RB69 DNA pol已发展成为用于研究B族DNA pols的模型酶。虽然所有B族DNA pol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 pol的天然抗药性和疱疹DNA pol的药物敏感性的分子机制仍然未知。在本研究中，我们构建了在DNA pol中包含G466S，Y460F，G466S / Y460F，P469S和V475W突变的T4噬菌体。这些氨基酸取代将药物敏感性疱疹家族DNA pols中发现的残基替换为耐药T4 DNA pol中的残基。我们调查了表达工程突变DNA pols的T4噬菌体是否对抗病毒药膦酰乙酸（PAA）敏感并在体内进行了表征噬菌体DNA pols的复制保真度。我们发现，G466S取代略微增加了PAA敏感性，而Y460F取代赋予了抗性。表达双突变体G466S / Y460F DNA pol的噬菌体对PAA的敏感性更高。V475W T4 DNA pol对PAA高度敏感，就像V478W RB69 DNA pol一样。但是，DNA复制受到严重损害，导致选择了表达更健壮的DNA pol的噬菌体，这些pol具有很强的复制DNA的能力，并含有抑制PAA敏感性的其他氨基酸取代。在表达PAA敏感的DNA pols的所有突变噬菌体中观察到复制保真度降低。这些观察结果表明PAA的敏感性和保真度在可以在不同环境中复制DNA的DNA pols中是平衡的。