Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase delta.
Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase delta.
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DOI：
10.1016/s0021-9258(18)52476-5
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发表时间：
1991-01
期刊：
影响因子：
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通讯作者：
Suk-Hee Lee;Ann D. Kwong;Zhen-Qiang Pan;J. Hurwitz
中科院分区：
文献类型：
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作者：
Suk-Hee Lee;Ann D. Kwong;Zhen-Qiang Pan;J. Hurwitz
激活子1（A1）是一种多蛋白复合物，其对于增殖细胞核抗原（PCNA）依赖性DNA聚合酶δ（pol δ）活性和SV 40二聚酶复制系统中的有效体外DNA合成是必需的。在这份报告中，我们描述了A1的HeLa细胞质提取物的分离。在含有PCNA、单链DNA结合蛋白（SSB）和ATP的反应中，A1刺激单一引发的phi X174 DNA或（dA）4500. oligo（dT）12-18中的pol δ活性。使用该测定，A1已被广泛纯化。纯化的制剂含有145、40、38、37和36.5 kDa的五个离散亚基。ATP水解为ADP和Pi对于A1依赖的pol δ活性是必不可少的，并且我们已经表明A1含有由DNA刺激的内在ATP酶。DNA依赖的ATP水解可被PCNA刺激，并进一步被PCNA和人单链DNA结合蛋白激活。用（dA）4500.oligo（dT）12-18观察到这些刺激作用，但单独用每种聚脱氧核苷酸未检测到。此外，A1与（dA）4500.oligo（dT）12-18形成复合物，该复合物可以通过硝酸纤维素结合来测量。通过该方法没有检测到与（dA）4500或单独的oligo（dT）12-18的复合物。数据还表明，A1，与PCNA，作为一个引物识别因子的聚合酶δ的功能，提高其利用低水平的引物末端的能力，但它不增加的DNA产物的大小。A1也显着减少了所需的多引物DNA上的pol δ活性的PCNA的量，表明PCNA与A1在引物末端相互作用。A1的这些多重作用与Tsurimoto和Stillman（Tsurimoto，T.，Stillman，B.等人（1990）Proc. Acad. Sci. U.S.A.87，1023-1027）。
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).