The ATP-activated hexameric helicase of bacteriophage T4 (gp41) forms a stable primosome with a single subunit of T4-coded primase (gp61)
The ATP-activated hexameric helicase of bacteriophage T4 (gp41) forms a stable primosome with a single subunit of T4-coded primase (gp61)
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DOI：
10.1074/jbc.271.32.19625
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发表时间：
1996-08-09
影响因子：
4.8
通讯作者：
vonHippel, PH
中科院分区：
文献类型：
--
作者：
Dong, F;vonHippel, PH
我们研究了由其解旋酶、引发酶和DNA成分组成的噬菌体T4编码的DNA复制（延伸）复合物的引发体亚组件的形成。此前，我们已经表明，在生理蛋白质（和盐）浓度下，T4解旋酶（gp41）在溶液中以稳定的单体 - 二聚体平衡存在，并且在通过ATP（或GTP）结合激活时形成六聚体（Dong，F.，Gogol，E. P.，和von Hippel，P. H.（1995）J. Biol. Chem. 270，7462 - 7473）。在此我们报道，在相同条件下，T4引发酶（gp61）在溶液中是单体，并且ATP激活的解旋酶在合适的DNA模板上与单个gp61引发酶分子结合以重建稳定的引发体。我们表明：（i）单独的gp41解旋酶不会与DNA模板形成稳定的复合物，尽管这种解旋酶本身能够沿着单链DNA进行适度的持续性ATP驱动的移位（Young，M. C.，Schultz，D. E.，Ring，D.，和von Hippel，P. H.（1994）J. Mol. Biol. 235，1447 - 1458）；（ii）单独的引发酶确实与DNA形成稳定的复合物；（iii）在ATP或GTP存在的情况下，解旋酶能够与引发酶 - DNA复合物结合以形成稳定的三元复合物；（iv）该复合物由六个解旋酶亚基和一个引发酶亚基组成；（v）在NTP裂解和水解产物解离后，重建的引发体至少在10到20分钟内是稳定的。这些结果强烈表明，功能性的T4 DNA复制引发体由结合到DNA的整合的6∶1解旋酶 - 引发酶复合物组成，并且ATP激活的解旋酶六聚体在持续性DNA复制过程中保持完整。
We have examined the formation of the primosome subassembly of the bacteriophage T4-coded DNA replication (elongation) complex from its helicase, primase, and DNA components, Previously, we had shown that the T4 helicase (gp41) exists in solution in a stable monomer dimer equilibrium at physiological protein (and salt) concentrations and forms a hexamer upon activation by ATP (or GTP) binding (Dong, F., Gogol, E. P., and von Hippel, P. H. (1995) J. Biol. Chem. 270, 7462-7473), Here we report that the T4 primase (gp61) is a monomer in solution under the same conditions, and that the ATP-activated helicase binds to a single gp61 primase molecule on appropriate DNA templates to reconstitute a stable primosome. We show that: (i) the gp41 helicase alone does not form a stable complex with DNA templates, although this helicase by itself can carry out moderately processive ATP-driven translocation along single-stranded DNA (Young, M. C., Schultz, D. E., Ring, D., and von Hippel, P. H. (1994) J. Mel. Biol. 235, 1447-1458); (ii) the primase alone does form a stable complex with DNA; (iii) the helicase can bind to the primase-DNA complex in the presence of ATP or GTP to form a stable ternary complex; (iv) this complex consists of six helicase subunits and one primase subunit; and (v) the reconstituted primosome is stable for at least 10 to 20 min after NTP cleavage and dissociation of the hydrolysis products, These results strongly suggest that the functional T4 DNA replication primosome consists of an integrated 6:1 helicase-primase complex bound to DNA, and that the ATP-activated helicase hexamer remains intact throughout the processive DNA replication process.