RAPID ASSEMBLY OF THE BACTERIOPHAGE-T4 CORE REPLICATION COMPLEX ON A LINEAR PRIMER TEMPLATE CONSTRUCT
RAPID ASSEMBLY OF THE BACTERIOPHAGE-T4 CORE REPLICATION COMPLEX ON A LINEAR PRIMER TEMPLATE CONSTRUCT
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DOI：
10.1073/pnas.90.22.10881
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发表时间：
1993-11-15
影响因子：
11.1
通讯作者：
BENKOVIC, SJ
中科院分区：
文献类型：
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作者：
KABOORD, BF;BENKOVIC, SJ
通过噬菌体T4在引发的DNA底物上进行DNA合成需要组装由T4 DNA聚合酶、单链结合蛋白（32蛋白）和辅助蛋白44/62和45组成的核心复制复合物。在本文中，我们证明了成功的组装，这个核心复合物上的短线性引物/模板系统的辅助蛋白的水平相当于引物3'端的浓度。这种组装的关键是链霉亲和素分子通过掺入模板链中的生物素部分结合在DNA底物的每一端。链霉抗生物素蛋白用于封闭引物/模板的末端，从而防止辅助蛋白从组装位点移位以及它们随后从引物/模板的末端解离。在这种底物上的复合物组装需要ATP以及44/62和45蛋白的存在。在我们的系统中，组装完整的酶当量复合物所需的时间几乎等于2秒。
DNA synthesis on a primed DNA substrate by bacteriophage T4 requires the assembly of a core replication complex consisting of the T4 DNA polymerase, a single-stranded binding protein (32 protein), and the accessory proteins 44/62 and 45. In this paper, we demonstrate the successful assembly of this core complex on a short linear primer/template system at levels of accessory proteins equivalent to the concentration of primer 3' ends. The key to this assembly is the presence of streptavidin molecules bound at each end of the DNA substrate via biotin moieties incorporated into the template strand. Streptavidin serves to block the ends of the primer/template, thus preventing translocation of the accessory proteins away from the site of assembly and their subsequent dissociation from the ends of the primer/template. Complex assembly on this substrate requires ATP and the presence of both the 44/62 and 45 proteins. The time required for assembly of a full enzyme equivalent of complex in our system is almost-equal-to 2 s.