Liu Y, Haggård-Ljungquist E
Department of Genetics, Stockholm University, Sweden.
Virology. 1996 Feb 1;216(1):158-64. doi: 10.1006/viro.1996.0042.
The A protein of bacteriophage P2 initiates DNA replication by a single-stranded cut at the origin, and the DNA replication proceeds unidirectionally by a modified rolling circle type of replication. The P2 A protein belongs to a family of proteins involved in the initiation of rolling circle DNA replication, and the prototype for this family is the well-characterized A protein of phage phi X174. One of the common motifs of this family contains two conserved tyrosine residues, which have been shown to be able to alternate in catalyzing the cleavage as well as joining reactions in the phi X174 A protein. We investigated the role of the conserved tyrosine residues in P2 A protein by in vitro mutagenesis. Only one of the two conserved tyrosine residues was found to be involved in the cleavage reaction. The tyrosine residue dispensable for cleavage and ligation is, however, required at some other stage of the P2 growth cycle, since viable recombinants containing this mutation could not be obtained. The sequence requirements for cleavage of the target site were analyzed with a set of oligonucleotides having single base alterations in the nick region, and the results indicate that only five core nucleotides need to be conserved for efficient cleavage.
噬菌体P2的A蛋白通过在复制起点处进行单链切割来启动DNA复制，DNA复制通过一种改良的滚环式复制单向进行。P2 A蛋白属于参与滚环DNA复制起始的蛋白家族，该家族的原型是特征明确的噬菌体φX174的A蛋白。这个家族的一个共同基序包含两个保守的酪氨酸残基，已证明它们能够交替催化φX174 A蛋白中的切割和连接反应。我们通过体外诱变研究了保守酪氨酸残基在P2 A蛋白中的作用。发现两个保守酪氨酸残基中只有一个参与切割反应。然而，对于切割和连接可 dispensable 的酪氨酸残基在P2生长周期的其他某个阶段是必需的，因为无法获得含有此突变的活重组体。用一组在切口区域具有单碱基改变的寡核苷酸分析了靶位点切割的序列要求，结果表明只需五个核心核苷酸保守即可有效切割。
