Kato M, Frick D N, Lee J, Tabor S, Richardson C C, Ellenberger T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 2001 Jun 15;276(24):21809-20. doi: 10.1074/jbc.M101470200. Epub 2001 Mar 28.
The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them. Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase.
复制叉的后随链最初是作为短冈崎片段被复制的，这些片段由两种依赖模板的酶的协同活性产生，一种是合成RNA引物的引发酶，另一种是延长引物的DNA聚合酶。噬菌体T7的基因4编码一种双功能引发酶-解旋酶，它组装成具有DNA解旋和引物合成活性的环状六聚体。T7 DNA聚合酶利用RNA引物也需要引发酶。目前尚不清楚引发酶-解旋酶六聚体中有多少亚基直接参与DNA合成的引发。为了确定T7 DNA聚合酶利用RNA引物的最低要求，我们构建了一种改变的基因4蛋白，它不能形成功能性六聚体，因此缺乏可检测到的DNA解旋活性。值得注意的是，这种单体引发酶很容易在单链模板上引发T7 DNA聚合酶进行DNA合成。单体基因4蛋白与T7 DNA聚合酶形成一种特异性的稳定复合物，从而将RNA引物递送至聚合酶以启动DNA合成。这些结果表明，引发酶-解旋酶六聚体的单个亚基包含引物合成以及T7 DNA聚合酶利用引物所需的所有残基。
