Roth M J, Brown D R, Hurwitz J
J Biol Chem. 1984 Aug 25;259(16):10556-68.
In the preceeding paper (Brown, D. R., Roth, M. J., Reinberg, D., and Hurwitz, J. (1984) J. Biol. Chem. 259, 10545-10555), it was shown that following bacteriophage phi X174 (phi X) DNA synthesis in vitro using purified proteins, the phi X A protein could be detected covalently linked to nascent 32P-labeled DNA. This phi X A protein-[32P]DNA complex was the product of the reinitiation reaction. The phi X A protein-[32P]DNA complex could be trapped as a protein-32P-oligonucleotide complex by the inclusion of ddGTP in reaction mixtures. In this report, the structure of the phi X A protein-32P-oligonucleotide complex has been analyzed. The DNA sequence of the oligonucleotide bound to the phi X A protein has been determined and shown to be homologous to the phi X (+) strand sequence immediately adjacent (3') to the replication origin. The phi X A protein was directly linked to the 5' position of a dAMP residue of the oligonucleotide; this residue corresponded to position 4306 of the phi X DNA sequence. The phi X A protein-32P-oligonucleotide complex was exhaustively digested with either trypsin or proteinase K and the 32P-labeled proteolytic fragments were analyzed. Each protease yielded two different 32P-labeled peptides in approximately equimolar ratios. The two 32P-labeled peptides formed after digestion with trypsin (designated T1 and T2) and with proteinase K (designated PK1 and PK2) were isolated and characterized. Digestion of peptide T1 with proteinase K yielded a product which co-migrated with peptide PK2. In contrast, peptide T2 was unaffected by digestion with proteinase K. These results suggest that the phi X A protein contains two active sites that are each capable of binding covalently to DNA. The peptide-mononucleotide complexes T1-[32P]pdA and T2-[32P]pdA were isolated and subjected to acid hydrolysis in 6.0 N HCl. In each case, the major 32P-labeled products were identified as [32P] phosphotyrosine and [32P]Pi. This indicates that each active site of the phi X A protein participates in a phosphodiester linkage between a tyrosyl moiety of the protein and the 5' position of dAMP.
在前一篇论文（Brown, D. R., Roth, M. J., Reinberg, D., and Hurwitz, J. (1984) J. Biol. Chem. 259, 10545 - 10555）中，已表明在体外使用纯化蛋白进行噬菌体φX174（φX）DNA合成后，可检测到φX A蛋白与新生的32P标记DNA共价连接。这种φX A蛋白 - [32P]DNA复合物是重新起始反应的产物。通过在反应混合物中加入ddGTP，φX A蛋白 - [32P]DNA复合物可被捕获为蛋白 - 32P - 寡核苷酸复合物。在本报告中，对φX A蛋白 - 32P - 寡核苷酸复合物的结构进行了分析。已确定与φX A蛋白结合的寡核苷酸的DNA序列，并显示其与紧邻复制起点（3'端）的φX（+）链序列同源。φX A蛋白直接与寡核苷酸的一个dAMP残基的5'位置相连；该残基对应于φX DNA序列的第4306位。用胰蛋白酶或蛋白酶K对φX A蛋白 - 32P - 寡核苷酸复合物进行彻底消化，并分析32P标记的蛋白水解片段。每种蛋白酶均产生两种摩尔比大致相等的不同32P标记肽段。分离并鉴定了用胰蛋白酶消化后形成的两种32P标记肽段（分别命名为T1和T2）以及用蛋白酶K消化后形成的肽段（分别命名为PK1和PK2）。用蛋白酶K消化肽段T1产生的产物与肽段PK2迁移率相同。相反，肽段T2不受蛋白酶K消化的影响。这些结果表明，φX A蛋白含有两个活性位点，每个活性位点都能够与DNA共价结合。分离出肽段单核苷酸复合物T1 - [32P]pdA和T2 - [32P]pdA，并在6.0 N HCl中进行酸水解。在每种情况下，主要的32P标记产物均被鉴定为[32P]磷酸酪氨酸和[32P]Pi。这表明φX A蛋白的每个活性位点都参与了蛋白质的酪氨酰部分与dAMP的5'位置之间的磷酸二酯键形成。
