Architecture of the active DNA polymerase δ• proliferating cell nuclear antigen•template-primer complex
Architecture of the active DNA polymerase δ• proliferating cell nuclear antigen•template-primer complex
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DOI：
10.1074/jbc.274.28.19862
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发表时间：
1999-07-09
影响因子：
4.8
通讯作者：
Fisher, PA
中科院分区：
文献类型：
--
作者：
Mozzherin, DJ;Tan, CK;Fisher, PA
研究了DNA依赖性DNA聚合酶δ（pol δ）增殖细胞核抗原（PCNA）DNA复合物组分的相对位置。我们已经表明，pol δ掺入核苷酸接近模板生物素-链霉亲和素复合物位于5'（下游）的复制复合物在存在或不存在的PCNA。位于模板3 '端的生物素链霉亲和素复合物与15-mer引物（复制复合物的上游）几乎完全（95%）抑制pol δ催化的PCNA依赖性合成，但仅部分抑制19-mer引物。使用任一引物，不依赖于PCNA的合成不受生物素链霉亲和素复合物的影响。用不同长度的引物对结果进行定量表明，pol δ与紧邻3 ′-OH引物末端的双链体DNA的8至10个核苷酸相互作用。利用UV光交联，我们确定pol δ的125-kDa亚基，而不是50-kDa亚基，与底物寡核苷酸的光敏残基相互作用，相互作用显然通过p125的C末端发生。基于这些结果，我们的结论是PCNA位于DNA合成过程中聚合复合物中pol δ的“后面”，（二亚基形式）直接与DNA相互作用。提出了酶活性复合物的详细模型。
The relative positions of components of the DNA-dependent DNA polymerase delta (pol delta) proliferating cell nuclear antigen (PCNA) DNA complex were studied. We have shown that pol delta incorporates nucleotides close to a template biotin-streptavidin complex located 5' (downstream) to the replicating complex in the presence or absence of PCNA. PCNA-dependent synthesis catalyzed by pol delta was nearly totally (95%) inhibited by a biotin streptavidin complex located at the 3'-end of a template with a 15-mer primer (upstream of the replicating complex), but was only partially inhibited with a 19-mer primer. With either primer, PCNA-independent synthesis was not affected by the biotin streptavidin complex. Quantification of results with primers of varying length suggested that pol delta interacts with between 8 and 10 nucleotides of duplex DNA immediately proximal to the 3'-OH primer terminus. Using UV photocross-linking, we determined that the 125-kDa subunit of pol delta, but not the 50-kDa subunit, interacted with a photosensitive residue of a substrate oligonucleotide, Interaction apparently takes place through the C terminus of p125, Based on these results, we conclude that PCNA is located "behind" pol delta in the polymerization complex during DNA synthesis and that only the large subunit of pol delta (two-subunit form) interacts directly with DNA. A detailed model of the enzymatically active complex is proposed.