从HeLa细胞中纯化与DNA聚合酶α相关的引发酶活性。

Purification of a primase activity associated with DNA polymerase alpha from HeLa cells.

作者信息

Gronostajski R M, Field J, Hurwitz J

出版信息

J Biol Chem. 1984 Aug 10;259(15):9479-86.

Abstract

Highly purified preparations of eukaryotic DNA polymerase alpha have been shown to contain primase activity (Kaguni, L.S., Rossignol, J-M., Conaway, R.C. Banks, G.R., and Lehman, I.R. (1983) J. Biol. Chem. 258, 9037-9039; Yagura, T., Kozu, T., and Seno, T. (1982) J. Biol. Chem. 257, 11121-11127; Shioda, M., Nelson, E.M., Bayne, M.L., and Benbow, R.M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7209-7213). We have investigated the de novo synthesis of DNA by a primase-DNA polymerase alpha preparation isolated from human HeLa cells using the synthetic homopolymers poly(dT) and poly(dC) as templates. In the presence of poly(dT), synthesis of poly(dA) required ATP in addition to dATP while synthesis of poly(dG) in the presence of poly(dC) required GTP in addition to dGTP. The primase activity required a much lower GTP concentration (Km = 0.1 mM) than ATP (Km = 0.8 mM) for the synthesis of DNA. Guanosine 5'-O-(3-thiotriphosphate), 5'-guanylyl-beta, gamma-imidodiphosphate, and 5'-guanylyl methylenediphosphonate substituted for GTP but the corresponding ATP analogues did not substitute for ATP. Furthermore, ATP and ATP analogues inhibited the GTP-dependent reaction while GTP and GTP analogues inhibited the ATP-dependent reaction. DNase treatment of products labeled with [alpha-32P] GTP revealed that an RNA oligomer was covalently linked to newly synthesized DNA. Alkaline hydrolysis of these products yielded GMP and pppGp, indicating that the primer was initiated with GTP. Alkaline hydrolysis of [alpha-32P]dGTP-labeled products yielded 2'- and 3'-GMP showing that DNA chains are covalently linked to the 3' ends of RNA chains. The primase activity could not be separated from DNA polymerase alpha through a 200-fold enrichment involving phosphocellulose, DNA-cellulose, hydroxylapatite, DEAE-cellulose and glycerol gradient purification steps. However, primase activity was found to be less stable than DNA polymerase alpha activity under a variety of conditions.

摘要

高度纯化的真核生物DNA聚合酶α制剂已被证明含有引发酶活性（卡古尼，L.S.，罗西尼奥尔，J - M.，康纳韦，R.C.，班克斯，G.R.，和莱曼，I.R.（1983年）《生物化学杂志》258，9037 - 9039；矢仓，T.，小津，T.，和濑野，T.（1982年）《生物化学杂志》257，11121 - 11127；盐田，M.，纳尔逊，E.M.，贝恩，M.L.，和本博，R.M.（1982年）《美国国家科学院院刊》79，7209 - 7213）。我们使用合成均聚物聚（dT）和聚（dC）作为模板，研究了从人宫颈癌细胞系HeLa细胞中分离出的引发酶 - DNA聚合酶α制剂对DNA的从头合成。在聚（dT）存在的情况下，聚（dA）的合成除了dATP外还需要ATP，而在聚（dC）存在的情况下聚（dG）的合成除了dGTP外还需要GTP。引发酶活性在DNA合成中所需的GTP浓度（Km = 0.1 mM）比ATP（Km = 0.8 mM）低得多。5'-O-（3-硫代三磷酸）鸟苷、5'-鸟苷酰-β，γ-亚氨基二磷酸和5'-鸟苷酰亚甲基二磷酸可替代GTP，但相应的ATP类似物不能替代ATP。此外，ATP和ATP类似物抑制GTP依赖性反应，而GTP和GTP类似物抑制ATP依赖性反应。用[α-32P]GTP标记的产物经DNase处理后显示，一个RNA寡聚物与新合成的DNA共价连接。这些产物的碱性水解产生GMP和pppGp，表明引物是以GTP起始的。用[α-32P]dGTP标记的产物经碱性水解产生2'-和3'-GMP，表明DNA链与RNA链的3'末端共价连接。经过磷酸纤维素、DNA纤维素、羟基磷灰石、DEAE纤维素和甘油梯度纯化步骤的200倍富集，引发酶活性仍无法与DNA聚合酶α分离。然而，发现在各种条件下引发酶活性比DNA聚合酶α活性更不稳定。