An auxiliary protein for DNA polymerase-delta from fetal calf thymus.
An auxiliary protein for DNA polymerase-delta from fetal calf thymus.
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DOI：
10.1016/s0021-9258(18)67240-0
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发表时间：
1986-09
期刊：
影响因子：
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通讯作者：
C. Tan;C. Castillo;A. So;K. Downey
中科院分区：
文献类型：
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作者：
C. Tan;C. Castillo;A. So;K. Downey
从同一组织中纯化了一种影响小牛胸腺DNA聚合酶-δ利用含有长链单链模板的模板/引物的能力的辅助蛋白。辅助蛋白在DEAE-纤维素和苯基-琼脂糖色谱上与DNA聚合酶-δ共洗脱，但在磷酸纤维素色谱上与聚合酶分离。辅助蛋白的物理和功能特性与大肠杆菌DNA聚合酶III全酶的β亚基非常相似。从沉降系数5.0 s和斯托克斯半径36.5 A计算出分子量为75，000。在十二烷基硫酸钠凝胶电泳上看到一条37，000道尔顿的单一条带，表明该蛋白质以相同亚基的二聚体形式存在。纯化的蛋白质没有可检测的DNA聚合酶、引发酶、ATP酶或核酸酶活性。DNA聚合酶-δ复制带缺口双链体DNA的能力相对不受辅助蛋白的影响，然而，需要复制具有低引物/模板比的模板，例如聚（dA）/寡聚（dT）（20：1）、引发的M13 DNA和变性的小牛胸腺DNA。该辅助蛋白对DNA聚合酶-δ具有特异性，对小牛胸腺DNA聚合酶-α或大肠杆菌Klenow片段的活性没有影响。coliDNA聚合酶I与引物均聚物模板。虽然辅助蛋白不与单链或双链DNA结合，但它确实增加了DNA聚合酶-δ与poly（dA）/oligo（dT）的结合，表明辅助蛋白在模板/引物存在下与聚合酶相互作用，稳定聚合酶-模板/引物复合物。
An auxiliary protein which affects the ability of calf thymus DNA polymerase-delta to utilize template/primers containing long stretches of single-stranded template has been purified to homogeneity from the same tissue. The auxiliary protein coelutes with DNA polymerase-delta on DEAE-cellulose and phenyl-agarose chromatography but is separated from the polymerase on phosphocellulose chromatography. The physical and functional properties of the auxiliary protein strongly resemble those of the beta subunit of Escherichia coli DNA polymerase III holoenzyme. A molecular weight of 75,000 has been calculated from a sedimentation coefficient of 5.0 s and a Stokes radius of 36.5 A. A single band of 37,000 daltons is seen on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein exists as a dimer of identical subunits. The purified protein has no detectable DNA polymerase, primase, ATPase, or nuclease activity. The ability of DNA polymerase-delta to replicate gapped duplex DNA is relatively unaffected by the presence of the auxiliary protein, however, it is required to replicate templates with low primer/template ratios, e.g. poly(dA)/oligo(dT) (20:1), primed M13 DNA, and denatured calf thymus DNA. The auxiliary protein is specific for DNA polymerase-delta; it has no effect on the activity of calf thymus DNA polymerase-alpha or the Klenow fragment of E. coli DNA polymerase I with primed homopolymer templates. Although the auxiliary protein does not bind to either single-stranded or double-stranded DNA, it does increase the binding of DNA polymerase-delta to poly(dA)/oligo(dT), suggesting that the auxiliary protein interacts with the polymerase in the presence of template/primer, stabilizing the polymerase-template/primer complex.