Suzuki M, Tamiya-Koizumi K, Takemura M, Furuta K, Izuta S, Savoysky E, Miura A, Yoshida S
Laboratory of Cancer Cell Biology, Nagoya University School of Medicine.
J Biochem. 1996 Oct;120(4):766-72. doi: 10.1093/oxfordjournals.jbchem.a021477.
We have shown that calf thymus DNA polymerase alpha-DNA primase complex (pol alpha-primase) preferentially binds to pyrimidine-rich sequences and initiates RNA primer synthesis [Suzuki, M. et al. (1993) Biochemistry 32, 12782-12792]. Here we tested the association of pol alpha-primase with a guanine-rich DNA fragment (SVG, 30-mer) containing in vivo initiation sites of simian virus 40 DNA replication. While pyrimidine-rich fragment (CTPPS 1, 30-mer), that is a preferred sequence for calf thymus DNA primase, was well co-precipitated with pol alpha-primase using anti-pol alpha antibody, SVG was hardly precipitated under the same conditions. Competition studies in either gel-retardation assay or during de novo DNA synthesis by pol alpha-primase demonstrated that the interaction of pol alpha-primase with SVG was much weaker than that with CTPPS-1. On the other hand, replication protein-A (RP-A) could bind SVG, although less efficiently than CTPPS 1. After preincubation with RP-A, SVG could bind pol alpha-primase that was immobilized on Sepharose beads. The simian virus 40 large T antigen also enhanced association of SVG to pol alpha-primase, while Escherichia coli single-stranded DNA-binding protein did not. However, pol alpha-primase, bound to SVG in the presence of RP-A, failed to synthesize RNA primers. When SVG was extended 10 nucleotides at its 5'-end, pol alpha-primase synthesized trace amounts of RNA primers, and this activity was stimulated more than 10-fold by adding RP-A. These results suggest a new role for RP-A, i.e., as a molecular tether that allows pol alpha-primase to bind guanine-rich regions of DNA in order to initiate RNA primer synthesis.
我们已经表明，小牛胸腺DNA聚合酶α-DNA引发酶复合物（polα-引发酶）优先结合富含嘧啶的序列并启动RNA引物合成[铃木，M.等人（1993年）《生物化学》32卷，12782 - 12792页]。在此，我们测试了polα-引发酶与一个富含鸟嘌呤的DNA片段（SVG，30聚体）的结合情况，该片段包含猴病毒40 DNA复制的体内起始位点。虽然富含嘧啶的片段（CTPPS 1，30聚体）是小牛胸腺DNA引发酶的首选序列，使用抗polα抗体能与polα-引发酶很好地共沉淀，但在相同条件下SVG几乎不沉淀。在凝胶阻滞试验或polα-引发酶的从头DNA合成过程中的竞争研究表明，polα-引发酶与SVG的相互作用比与CTPPS - 1的相互作用弱得多。另一方面，复制蛋白A（RP - A）可以结合SVG，尽管效率比CTPPS 1低。与RP - A预孵育后，SVG可以结合固定在琼脂糖珠上的polα-引发酶。猴病毒40大T抗原也增强了SVG与polα-引发酶的结合，而大肠杆菌单链DNA结合蛋白则没有。然而，在RP - A存在下与SVG结合的polα-引发酶无法合成RNA引物。当SVG在其5'端延伸10个核苷酸时，polα-引发酶合成了微量的RNA引物，并且通过添加RP - A这种活性被刺激了10倍以上。这些结果表明了RP - A的一个新作用，即作为一种分子系绳，使polα-引发酶能够结合DNA的富含鸟嘌呤区域以启动RNA引物合成。
