Rodríguez Irene, Lázaro José M, Salas Margarita, de Vega Miguel
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Cantoblanco, E-28049 Madrid, Spain.
J Mol Biol. 2003 Jan 3;325(1):85-97. doi: 10.1016/s0022-2836(02)01130-0.
Bacteriophage phi29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as primer for initiation of DNA replication. By multiple sequence alignments of DNA polymerases from such a family, we have been able to identify two amino acid residues specifically conserved in the protein-priming subgroup of DNA polymerases, a phenylalanine contained in the (S/T)Lx(2)h motif, and a glutamate belonging to the Exo III motif. Here, we have studied the functional role of these residues in reactions that are specific for DNA polymerases that use a protein-primed DNA replication mechanism, by site-directed mutagenesis in the corresponding amino acid residues, Phe128 and Glu161 of phi29 DNA polymerase. Mutations introduced at residue Phe128 severely impaired the protein-primed replication capacity of the polymerase, being the interaction with the terminal protein (TP) moderately (mutant F128A) or severely (mutant F128Y) diminished. As a consequence, very few initiation products were obtained, and essentially no transition products were detected. Interestingly, phi29 DNA polymerase mutant F128Y showed a decreased binding affinity for short template DNA molecules. These results, together with the high degree of conservation of Phe128 residue among protein-primed DNA polymerases, suggest a functional role for this amino acid residue in making contacts with the TP during the first steps of genome replication and with DNA in the further replication steps.
噬菌体phi29编码一种依赖DNA的DNA聚合酶，它属于DNA聚合酶的真核生物类型（B家族）亚组，该亚组以蛋白质作为DNA复制起始的引物。通过对来自该家族的DNA聚合酶进行多序列比对，我们能够鉴定出在蛋白质引发的DNA聚合酶亚组中特异性保守的两个氨基酸残基，一个是位于(S/T)Lx(2)h基序中的苯丙氨酸，另一个是属于Exo III基序的谷氨酸。在此，我们通过对phi29 DNA聚合酶相应氨基酸残基Phe128和Glu161进行定点诱变，研究了这些残基在使用蛋白质引发DNA复制机制的DNA聚合酶特异性反应中的功能作用。在残基Phe128处引入的突变严重损害了聚合酶的蛋白质引发复制能力，与末端蛋白(TP)的相互作用中度（突变体F128A）或严重（突变体F128Y）减弱。结果，获得的起始产物很少，基本上没有检测到过渡产物。有趣的是，phi29 DNA聚合酶突变体F128Y对短模板DNA分子的结合亲和力降低。这些结果，连同蛋白质引发的DNA聚合酶中Phe128残基的高度保守性，表明该氨基酸残基在基因组复制的第一步与TP接触以及在进一步的复制步骤与DNA接触中具有功能作用。
