Lee S H, Kwong A D, Pan Z Q, Hurwitz J
Graduate Program in Molecular Biology, Sloan-Kettering Cancer Center, New York, New York 10021.
J Biol Chem. 1991 Jan 5;266(1):594-602.
Activator 1 (A1) is a multiprotein complex which is essential for proliferating cell nuclear antigen (PCNA)-dependent DNA polymerase delta (pol delta) activity and efficient in vitro DNA synthesis in the SV40 dipolymerase replication system. In this report, we describe the isolation of A1 from HeLa cytosolic extracts. A1 stimulated pol delta activity in singly primed phi X174 DNA or (dA)4500.oligo(dT)12-18 in reactions containing PCNA, single-stranded DNA binding protein (SSB), and ATP. Using this assay, A1 has been extensively purified. Purified preparations contained five discrete subunits of 145, 40, 38, 37, and 36.5 kDa. ATP hydrolysis to ADP and Pi is essential for A1-dependent pol delta activity, and we have shown that A1 contains an intrinsic ATPase which is stimulated by DNA. The DNA-dependent hydrolysis of ATP can be stimulated by PCNA and further activated by PCNA plus the human single-stranded DNA binding protein. These stimulatory effects were observed with (dA)4500.oligo(dT)12-18, but were not detected with each poly-deoxynucleotide alone. Furthermore, A1 formed a complex with (dA)4500.oligo(dT)12-18 which could be measured by nitrocellulose binding. No complex with (dA)4500 or oligo(dT)12-18 alone was detected by this procedure. Data are also presented which indicate that A1, in conjunction with PCNA, functions as a primer-recognition factor for pol delta, increasing its ability to utilize low levels of primer ends, but it does not increase the size of the DNA products. A1 also markedly reduced the amount of PCNA required for pol delta activity on a multiply primed DNA suggesting that PCNA interacts with A1 at the primer end. These multiple effects of A1 closely resemble the properties of the multisubunit protein RF-C described by Tsurimoto and Stillman (Tsurimoto, T., and Stillman, B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1023-1027).
激活因子1（A1）是一种多蛋白复合物，对于增殖细胞核抗原（PCNA）依赖性DNA聚合酶δ（pol δ）的活性以及SV40二聚体聚合酶复制系统中高效的体外DNA合成至关重要。在本报告中，我们描述了从HeLa细胞胞质提取物中分离A1的过程。在含有PCNA、单链DNA结合蛋白（SSB）和ATP的反应中，A1刺激了单引物φX174 DNA或（dA）4500·寡聚（dT）12 - 18中的pol δ活性。利用该检测方法，A1已被广泛纯化。纯化后的制剂包含五个不同的亚基，分子量分别为145、40、38、37和36.5 kDa。ATP水解为ADP和Pi对于依赖A1的pol δ活性至关重要，并且我们已表明A1含有一种受DNA刺激的内在ATP酶。DNA依赖性的ATP水解可被PCNA刺激，并被PCNA加上人单链DNA结合蛋白进一步激活。这些刺激作用在（dA）4500·寡聚（dT）12 - 18中观察到，但单独使用每种多脱氧核苷酸时未检测到。此外，A1与（dA）450·寡聚（dT）12 - 18形成了一种可通过硝酸纤维素结合来测量的复合物。通过该方法未检测到与单独的（dA）4500或寡聚（dT）12 - 18形成的复合物。还呈现了数据表明A1与PCNA一起作为pol δ的引物识别因子发挥作用，提高其利用低水平引物末端的能力，但不增加DNA产物的大小。A1还显著减少了在多引物DNA上pol δ活性所需的PCNA量，这表明PCNA在引物末端与A1相互作用。A1的这些多种作用与Tsurimoto和Stillman描述（Tsurimoto, T., and Stillman, B. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1023 - 1027）的多亚基蛋白RF - C的特性非常相似。
