Bravo A, Hermoso J M, Salas M
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Madrid, Spain.
Mol Gen Genet. 1994 Dec 1;245(5):529-36. doi: 10.1007/BF00282215.
Protein p6 of the Bacillus subtilis phage phi 29 is essential for in vivo viral DNA replication. This protein activates the initiation of phi 29 DNA replication in vitro by forming a multimeric nucleoprotein complex at the replication origins. The N-terminal region of protein p6 is involved in DNA binding, as shown by in vitro studies with p6 proteins altered by deletions or missense mutations. We report on the development of an in vivo functional assay for protein p6. This assay is based on the ability of protein p6-producing B. subtilis non-suppressor (su) cells to support growth of a phi 29 sus6 mutant phage. We have used this trans-complementation assay to investigate the effect on in vivo viral DNA synthesis of missense mutations introduced into the protein p6 N-terminal region. The alteration of lysine to alanine at position 2 resulted in a partially functional protein, whereas the replacement of arginine by alanine at position 6 gave rise to an inactive protein. These results indicate that arginine at position 6 is critical for the in vivo activity of protein p6. Our complementation system provides a useful genetic approach for the identification of functionally important amino acids in protein p6.
枯草芽孢杆菌噬菌体phi 29的蛋白质p6对体内病毒DNA复制至关重要。该蛋白质通过在复制起点形成多聚体核蛋白复合物，在体外激活phi 29 DNA复制的起始。如对因缺失或错义突变而改变的p6蛋白进行的体外研究所显示，蛋白质p6的N端区域参与DNA结合。我们报告了一种针对蛋白质p6的体内功能检测方法的开发。该检测基于产生蛋白质p6的枯草芽孢杆菌非抑制（su）细胞支持phi 29 sus6突变体噬菌体生长的能力。我们已使用这种反式互补检测方法来研究引入蛋白质p6 N端区域的错义突变对体内病毒DNA合成的影响。第2位赖氨酸变为丙氨酸导致蛋白质部分功能化，而第6位精氨酸被丙氨酸取代则产生无活性的蛋白质。这些结果表明，第6位的精氨酸对蛋白质p6的体内活性至关重要。我们的互补系统为鉴定蛋白质p6中功能重要的氨基酸提供了一种有用的遗传学方法。
