Mizushina Y, Matsukage A, Sakaguchi K
Department of Applied Biological Science, Science University of Tokyo, Noda, Chiba 278-8510, Japan.
Biochim Biophys Acta. 1998 May 27;1403(1):5-11. doi: 10.1016/s0167-4889(98)00027-5.
We reported previously [T. Horie, Y. Mizushina, M. Takemura, F. Sugawara, A. Matsukage, S. Yoshida, K. Sakaguchi, Int. J. Mol. Med., 1 (1998) 83-90.] that a 5'-monophosphate form (breMP) of bredinin, which has been used clinically as an immunosuppressive drug, selectively suppressed the activities of mammalian DNA polymerase alpha (pol. alpha) and beta (pol. beta). In a preliminary study of the action mode, for pol. beta, breMP acted by competing with, unexpectedly, not only the substrate but also with the template-primer. The mode might be attributable to the structure and function of pol. beta itself. We therefore investigated the biochemical inhibition mode of pol. beta in more detail by using two pol. beta fragments which were proteolytically separated into the template-primer-binding domain and the catalytic domain. BreMP inhibited only the catalytic activity of the catalytic domain fragment, and could not bind to the template-primer-binding domain fragment, suggesting that it directly competes with the substrate at its binding site of the catalytic domain, and indirectly, but simultaneously and competitively disturbs the template-primer incorporation into the template-primer-binding domain.
我们之前报道过[T. Horie, Y. Mizushina, M. Takemura, F. Sugawara, A. Matsukage, S. Yoshida, K. Sakaguchi, Int. J. Mol. Med., 1 (1998) 83 - 90.]，临床上用作免疫抑制药物的布累迪宁的5'-单磷酸形式（breMP）能选择性抑制哺乳动物DNA聚合酶α（pol.α）和β（pol.β）的活性。在作用模式的初步研究中，对于pol.β，breMP的作用方式出人意料，它不仅与底物竞争，还与模板引物竞争。这种模式可能归因于pol.β自身的结构和功能。因此，我们通过使用两个经蛋白酶水解分离成模板引物结合结构域和催化结构域的pol.β片段，更详细地研究了pol.β的生化抑制模式。BreMP仅抑制催化结构域片段的催化活性，且不能与模板引物结合结构域片段结合，这表明它在催化结构域的结合位点直接与底物竞争，并且间接但同时竞争性地干扰模板引物掺入模板引物结合结构域。
