LaDuca R J, Crute J J, McHenry C S, Bambara R A
J Biol Chem. 1986 Jun 5;261(16):7550-7.
We have previously demonstrated that the addition of a stoichiometric excess of the beta subunit of Escherichia coli DNA polymerase III holoenzyme to DNA polymerase III or holoenzyme itself can lead to an ATP-independent increase in the processivity of these enzyme forms (Crute, J. J., LaDuca, R. J., Johanson, K. O., McHenry, C. S., and Bambara, R. A. (1983) J. Biol. Chem. 258, 11344-11349). Here, we show that the beta subunit can interact directly with the catalytic core of the holoenzyme, DNA polymerase III, generating a new form of the enzyme with enhanced catalytic and processive capabilities. The addition of saturating levels of the beta subunit to the core DNA polymerase III enzyme results in as much as a 7-fold stimulation of synthetic activity. Two populations of DNA products were generated by the DNA polymerase III X beta enzyme complex. Short products resulting from the addition of 5-10 nucleotides/primer fragment were generated by DNA polymerase III in the presence and absence of added beta subunit. A second population of much longer products was generated only in beta-supplemented DNA polymerase III reactions. The DNA polymerase III-beta reaction was inhibited by single-stranded DNA binding protein and was unaffected by ATP, distinguishing it from the holoenzyme-catalyzed reaction. Complex formation of the DNA polymerase III core enzyme with beta increased the residence time of the enzyme on synthetic DNA templates. Our results demonstrate that the beta stimulation of DNA polymerase III can be attributed to a more efficient and highly processive elongation capability of the DNA polymerase III X beta complex. They also prove that at least part of beta's normal contribution to the DNA polymerase III holoenzyme reaction takes place through interaction with DNA polymerase III core enzyme components to produce the essential complex necessary for efficient elongation in vivo.
我们之前已经证明，向大肠杆菌DNA聚合酶III全酶或其本身添加化学计量过量的β亚基，可导致这些酶形式的持续合成能力在不依赖ATP的情况下增加（克鲁特，J. J.，拉杜卡，R. J.，约翰森，K. O.，麦克亨利，C. S.，和班巴拉，R. A.（1983年）《生物化学杂志》258，11344 - 11349）。在此，我们表明β亚基可直接与全酶的催化核心DNA聚合酶III相互作用，产生一种具有增强催化和持续合成能力的新酶形式。向核心DNA聚合酶III酶中添加饱和水平的β亚基可使合成活性提高多达7倍。DNA聚合酶III与β酶复合物产生了两类DNA产物。在添加和不添加β亚基的情况下，DNA聚合酶III都会产生因添加5 - 10个核苷酸/引物片段而形成的短产物。仅在添加了β亚基的DNA聚合酶III反应中会产生另一类长得多的产物。DNA聚合酶III - β反应受到单链DNA结合蛋白的抑制，且不受ATP影响，这使其有别于全酶催化的反应。DNA聚合酶III核心酶与β亚基的复合物形成增加了该酶在合成DNA模板上的停留时间。我们的结果表明，β亚基对DNA聚合酶III的促进作用可归因于DNA聚合酶III与β亚基复合物更高效且高度持续的延伸能力。这些结果还证明，β亚基对DNA聚合酶III全酶反应的正常贡献至少部分是通过与DNA聚合酶III核心酶组分相互作用来实现的，从而产生体内高效延伸所必需的关键复合物。
