Tsurimoto T, Stillman B
Cold Spring Harbor Laboratory, New York 11724.
J Biol Chem. 1991 Jan 25;266(3):1950-60.
Eukaryotic DNA polymerase delta and its accessory proteins are essential for SV40 DNA replication in vitro. A multi-subunit protein complex, replication factor C (RF-C), which is composed of subunits with apparent molecular weights of 140,000, 41,000, and 37,000, has primer/template binding and DNA-dependent ATPase activities. UV-cross-linking experiments demonstrated that the Mr = 140,000 subunit recognizes and binds to the primer-template DNA, whereas the Mr = 41,000 polypeptide binds ATP. Assembly of a replication complex at a primer-template junction has been studied in detail with synthetic, hairpin DNAs. Following glutaraldehyde fixation, a gel shift assay demonstrated that RF-C alone forms a weak binding complex with the hairpin DNA. Addition of ATP or its nonhydrolyzable analogue, ATP gamma S, increased specific binding to the DNA. Footprinting experiments revealed that RF-C recognizes the primer-template junction, covering 15 bases of the primer DNA from the 3'-end and 20 bases of the template DNA. Another replication factor, proliferating cell nuclear antigen (PCNA) binds to RF-C and the primer-template DNA forming a primer recognition complex and extends the protected region on the duplex DNA. This RF-C.PCNA complex has significant single-stranded DNA binding activity in addition to binding to a primer-template junction. However, addition of another replication factor, RF-A, completely blocked the nonspecific, single-stranded DNA binding by the RF-C.PCNA complex. RF-A therefore functions as a specificity factor for primer recognition. In the absence of RF-C, DNA polymerase delta (pol delta) and PCNA form a complex at the primer-template junction, protecting exactly the same site as the primer recognition complex. Addition of RF-C to this complex produced a higher order complex which is unstable unless its formation is coupled with translocation of pol delta. These results suggest that the sequential binding of RF-C, PCNA, and pol delta to a primer-template junction might directly account for the initiation of leading strand DNA synthesis at a replication origin. We demonstrate this directly in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1961-1968).
真核生物DNA聚合酶δ及其辅助蛋白对于体外SV40 DNA复制至关重要。一种多亚基蛋白复合物，复制因子C（RF-C），由表观分子量为140,000、41,000和37,000的亚基组成，具有引物/模板结合和依赖于DNA的ATP酶活性。紫外线交联实验表明，分子量为140,000的亚基识别并结合引物-模板DNA，而分子量为41,000的多肽结合ATP。已使用合成的发夹DNA详细研究了在引物-模板连接处复制复合物的组装。戊二醛固定后，凝胶迁移分析表明，单独的RF-C与发夹DNA形成弱结合复合物。添加ATP或其不可水解的类似物ATPγS可增加与DNA的特异性结合。足迹实验表明，RF-C识别引物-模板连接处，覆盖引物DNA 3'端的15个碱基和模板DNA的20个碱基。另一种复制因子，增殖细胞核抗原（PCNA）与RF-C和引物-模板DNA结合，形成引物识别复合物，并扩展双链DNA上的保护区域。这种RF-C.PCNA复合物除了结合引物-模板连接处外，还具有显著的单链DNA结合活性。然而，添加另一种复制因子RF-A完全阻断了RF-C.PCNA复合物的非特异性单链DNA结合。因此，RF-A作为引物识别的特异性因子发挥作用。在没有RF-C的情况下，DNA聚合酶δ（polδ）和PCNA在引物-模板连接处形成复合物，保护与引物识别复合物完全相同的确切位点。向该复合物中添加RF-C会产生更高阶的复合物，除非其形成与polδ的转位偶联，否则该复合物不稳定。这些结果表明，RF-C、PCNA和polδ依次与引物-模板连接处结合可能直接解释了复制起点处前导链DNA合成的起始。我们在随附的论文中直接证明了这一点（Tsurimoto，T.，和Stillman，B.（1991）J. Biol. Chem. 266，1961 - 1968）。
