Weisshart K, Förster H, Kremmer E, Schlott B, Grosse F, Nasheuer H P
Institut für Molekulare Biotechnologie e.V., Abteilung Biochemie, Beutenbergstrasse 11, D-07745 Jena, Germany.
J Biol Chem. 2000 Jun 9;275(23):17328-37. doi: 10.1074/jbc.M000717200.
DNA polymerase alpha-primase (pol-prim, consisting of p180-p68-p58-p48), and primase p58-p48 (prim(2)) synthesize short RNA primers on single-stranded DNA. In the SV40 DNA replication system, only pol-prim is able to start leading strand DNA replication that needs unwinding of double-stranded (ds) DNA prior to primer synthesis. At high concentrations, pol-prim and prim(2) indistinguishably reduce the unwinding of dsDNA by SV40 T antigen (Tag). RNA primer synthesis on ssDNA in the presence of replication protein A (RPA) and Tag has served as a model system to study the initiation of Okazaki fragments on the lagging strand in vitro. On ssDNA, Tag stimulates whereas RPA inhibits the initiation reaction of both enzymes. Tag reverses and even overcompensates the inhibition of primase by RPA. Physical binding of Tag to the primase subunits and RPA, respectively, is required for these activities. Each subunit of the primase complex, p58 and p48, performs physical contacts with Tag and RPA independently of p180 and p68. Using surface plasmon resonance, the dissociation constants of the Tag/pol-prim and Tag/primase interactions were 1.2 x 10(-8) m and 1.3 x 10(-8) m, respectively.
DNA聚合酶α-引发酶（pol-prim，由p180-p68-p58-p48组成）和引发酶p58-p48（prim(2)）在单链DNA上合成短RNA引物。在SV40 DNA复制系统中，只有pol-prim能够启动前导链DNA复制，该复制在引物合成之前需要解开双链（ds）DNA。在高浓度下，pol-prim和prim(2)可无差别地降低SV40 T抗原（Tag）介导的dsDNA解旋。在复制蛋白A（RPA）和Tag存在的情况下，在ssDNA上进行RNA引物合成已成为体外研究滞后链上冈崎片段起始的模型系统。在ssDNA上，Tag起促进作用，而RPA则抑制这两种酶的起始反应。Tag可逆转甚至过度补偿RPA对引发酶的抑制作用。Tag分别与引发酶亚基和RPA的物理结合是这些活性所必需的。引发酶复合物的每个亚基，p58和p48，独立于p180和p68与Tag和RPA进行物理接触。使用表面等离子体共振技术，Tag/pol-prim和Tag/引发酶相互作用的解离常数分别为1.2×10⁻⁸ m和1.3×10⁻⁸ m。
