van Mansfeld A D, van Teeffelen H A, Fluit A C, Baas P D, Jansz H S
Nucleic Acids Res. 1986 Feb 25;14(4):1845-61. doi: 10.1093/nar/14.4.1845.
Gene A protein of bacteriophage phi X174 plays a role as a site-specific endonuclease in the initiation and termination of phi X rolling circle DNA replication. To clarify the sequence requirements of this protein we have studied the cleavage of single-stranded restriction fragments from phi X and G4 viral DNAs using purified gene A protein. The results show that in both viral DNAs cleavage occurs at the origin and at one additional site which shows striking sequence homology with the origin region. During rolling circle replication the single-stranded viral DNA tail is covered with single-stranded DNA binding (SSB) protein. Therefore, we have also studied the effect of SSB on phi X gene A protein cleavage. In these conditions only single-stranded fragments containing the complete or almost complete origin region of 30 bases are cleaved, whereas cleavage at the additional sites of phi X or G4 viral DNAs does not occur. A model for termination of rolling circle replication which is based on these findings is presented. Finally, we present evidence that the second product of gene A, the A* protein, cleaves phi X viral DNA at the additional cleavage site in the presence of SSB, not only in vitro but also in vivo. The functional significance of this cleavage in vivo is discussed.
噬菌体φX174的基因A蛋白在φX滚环DNA复制的起始和终止过程中作为位点特异性内切核酸酶发挥作用。为了阐明该蛋白的序列要求，我们使用纯化的基因A蛋白研究了来自φX和G4病毒DNA的单链限制性片段的切割。结果表明，在两种病毒DNA中，切割均发生在起始位点和另一个与起始区域具有显著序列同源性的位点。在滚环复制过程中，单链病毒DNA尾巴被单链DNA结合（SSB）蛋白覆盖。因此，我们还研究了SSB对φX基因A蛋白切割的影响。在这些条件下，只有包含30个碱基的完整或几乎完整起始区域的单链片段被切割，而在φX或G4病毒DNA的其他位点则不发生切割。基于这些发现，我们提出了一个滚环复制终止的模型。最后，我们提供证据表明，基因A的第二个产物A*蛋白不仅在体外，而且在体内，在SSB存在的情况下，在额外的切割位点切割φX病毒DNA。我们讨论了这种切割在体内的功能意义。
