Mikhaĭlov V S, Voronova S N, Akopov S B, Kuldyev P K, Atrazhev A M
Mol Biol (Mosk). 1991 Jan-Feb;25(1):240-9.
Depending on the ionic environment the replicative complex of silkworm Bombyx mori, containing DNA polymerase alpha and primase, catalyzes on single-stranded DNA of phage M13 a NTP-dependent synthesis or elongation of preformed primers. In the presence of NTPs and dNTPs at conditions optimal for the NTP-dependent synthesis the replicative complex synthesizes on M13 DNA oligoribonucleotides of 9-11 residues, which serve as primers for polymerization of DNA. The length of RNA-primers synthesized by primase of the complex depends on concentration of dNTP but does not depend on activity of DNA polymerase alpha. During elongation of exogenic primers annealed to M13 DNA the complex is processive synthesizing DNA fragments of dozens residues without dissociation from the template. Double-stranded structures in DNA such as "hairpins" appear to be barriers for driving of the complex along the template and cause pauses in elongation. DNA-binding proteins the SSB of Escherichia coli or the p32 of phage T4 destabilize double-stranded regions in DNA and eliminate elongation pauses corresponding to these regions. The replicative complex is able to fill in single-stranded gaps in DNA completely and to perform slowly the synthesis with displacement of one of parent strands in duplexes via repeated cycles of binding to the primer-template, limited elongation and dissociation.
根据离子环境,家蚕(Bombyx mori)的复制复合体(包含DNA聚合酶α和引发酶)可催化在噬菌体M13的单链DNA上进行NTP依赖性的预形成引物的合成或延伸。在NTP依赖性合成的最佳条件下,存在NTP和dNTP时,复制复合体可在M13 DNA上合成9 - 11个残基的寡核糖核苷酸,这些寡核糖核苷酸可作为DNA聚合的引物。该复合体引发酶合成的RNA引物长度取决于dNTP的浓度,但不取决于DNA聚合酶α的活性。在外源引物与M13 DNA退火后的延伸过程中,该复合体具有持续性,可合成数十个残基的DNA片段而不与模板解离。DNA中的双链结构如“发夹”似乎是复合体沿模板移动的障碍,并导致延伸过程中的停顿。大肠杆菌的单链结合蛋白(SSB)或噬菌体T4的p32等DNA结合蛋白会使DNA中的双链区域不稳定,并消除与这些区域对应的延伸停顿。该复制复合体能够完全填补DNA中的单链缺口,并通过与引物 - 模板反复结合、有限延伸和解离的循环,缓慢地进行双链中一条亲代链被置换的合成。