Aybakan Esma, Kocagoz Tanil, Can Ozge
Department of Medical Biotechnology, Institute of Health Sciences, Acibadem Mehmet Ali Aydinlar University, 34752 Istanbul, Türkiye.
Department of Medical Microbiology, School of Medicine, Acibadem Mehmet Ali Aydinlar University, 34752 Istanbul, Türkiye.
Int J Mol Sci. 2025 Jan 18;26(2):789. doi: 10.3390/ijms26020789.
Gene II Protein (Gp2/P2) is a nicking enzyme of the M13 bacteriophage that plays a role in the DNA replication of the viral genome. P2 recognizes a specific sequence at the f1 replication origin and nicks one of the strands and starts replication. This study was conducted to address the limitations of previous experiments, improve methodologies, and precisely determine the biochemical activity conditions of the P2 enzyme in vitro. For these purposes, the gene encoding P2 was cloned in and expressed as a hybrid protein together with a green fluorescent protein (P2-GFP). P2-GFP was purified via metal affinity chromatography, and its nicking activity was determined by conversion of supercoiled DNA to open circular or linear forms. We discovered that, among the two loops of the f1 origin defined previously, P2 can recognize just the A1 loop. When a supercoiled plasmid containing the f1 origin was treated with P2-GFP, the plasmid was present in an open circular form, indicating that a nick was created on only one of the strands. However, when the A1 loop sequence was inserted into the 3' ends of both strands by cloning a PCR product obtained by primers with the A1 loop sequence, the plasmid was linearized by treatment with P2-GFP, indicating that nicks were created on both strands. Certain infectious diseases are caused by single-stranded DNA viruses, and some of them have specific nicking enzymes that enable strand displacement and free 3' end of a single strand that works as a primer for their replication mechanisms like M13 bacteriophages, such as parvovirus B19. Despite there being different host viruses such as bacteria and humans, their DNA replication mechanisms are very similar in this concept. Investigating the features of the P2-nicking enzyme may deepen the understanding of human pathogenic single-stranded viruses and facilitate the development of drugs that inhibit viral replication.
基因II蛋白（Gp2/P2）是M13噬菌体的一种切口酶，在病毒基因组的DNA复制中发挥作用。P2识别f1复制起点处的特定序列，在其中一条链上造成切口并启动复制。本研究旨在解决先前实验的局限性，改进方法，并精确确定P2酶在体外的生化活性条件。为此，编码P2的基因被克隆并与绿色荧光蛋白一起表达为融合蛋白（P2-GFP）。P2-GFP通过金属亲和层析进行纯化，其切口活性通过将超螺旋DNA转化为开环或线性形式来确定。我们发现，在先前定义的f1起点的两个环中，P2只能识别A1环。当用P2-GFP处理含有f1起点的超螺旋质粒时，质粒呈现开环形式，表明仅在一条链上产生了切口。然而，当通过克隆由带有A1环序列的引物获得的PCR产物，将A1环序列插入两条链的3'端时，用P2-GFP处理后质粒被线性化，表明两条链上都产生了切口。某些传染病由单链DNA病毒引起，其中一些具有特定的切口酶，能够实现链置换并产生单链的游离3'端，该游离3'端可作为其复制机制（如M13噬菌体，如细小病毒B19）的引物。尽管存在细菌和人类等不同的宿主病毒，但在这一概念上它们的DNA复制机制非常相似。研究P2切口酶的特性可能会加深对人类致病性单链病毒的理解，并促进抑制病毒复制的药物的开发。
