Mizushina Y, Yoshida S, Matsukage A, Sakaguchi K
Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, Japan.
Biochim Biophys Acta. 1997 Oct 20;1336(3):509-21. doi: 10.1016/s0304-4165(97)00067-6.
We found previously that long-chain fatty acids could inhibit eukaryotic DNA polymerase activities in vitro [1,2]. The purpose of the present study was to investigate the mode of this inhibition in greater detail. Among the C18 to C24 fatty acids examined, the strongest inhibitor was a C24 fatty acid, nervonic acid (NA), and the weakest was a C18 fatty acid, linoleic acid (LA). We analyzed the inhibitory effect of these two fatty acids and their modes of action. For DNA polymerase beta (pol. beta), NA acted by competing with both the substrate- and template-primer, but for DNA polymerase alpha (pol. alpha) or human immunodeficiency virus type 1 reverse transcriptase (HIV-1 reverse transcriptase or HIV-RT), NA acted non-competitively. NA-binding to pol. beta could be stopped with a non-ionic detergent, but the binding to pol. alpha or HIV-RT could not. The inhibition mode of LA showed the same characteristics, except that the minimum inhibitory dose of the longer chain was much lower. We also tested the effects of NA and LA using pol. beta and its proteolytic fragments, as described by Kumar et al. [3,4]. Both of the fatty acids were found to bind to the 8 kDa DNA-binding domain fragment, and to suppress binding to the template-primer DNA. We found that 10,000 times more of either fatty acid was required for it to bind to the 31 kDa catalytic domain or inhibit the DNA polymerase activity. The possible modes of inhibition by these long-chain fatty acids are discussed, based on the present findings.
我们之前发现长链脂肪酸在体外可抑制真核生物DNA聚合酶的活性[1,2]。本研究的目的是更详细地探究这种抑制作用的方式。在所检测的C18至C24脂肪酸中，最强的抑制剂是C24脂肪酸神经酸（NA），最弱的是C18脂肪酸亚油酸（LA）。我们分析了这两种脂肪酸的抑制作用及其作用方式。对于DNA聚合酶β（pol.β），NA通过与底物及模板引物竞争起作用，但对于DNA聚合酶α（pol.α）或人类免疫缺陷病毒1型逆转录酶（HIV - 1逆转录酶或HIV - RT），NA起非竞争性作用。NA与pol.β的结合可用非离子去污剂阻断，但与pol.α或HIV - RT的结合则不能。LA的抑制模式显示出相同的特征，只是较长链脂肪酸的最小抑制剂量要低得多。我们还按照Kumar等人[3,4]所述，用pol.β及其蛋白水解片段测试了NA和LA的作用。发现这两种脂肪酸都能与8 kDa的DNA结合结构域片段结合，并抑制与模板引物DNA的结合。我们发现，这两种脂肪酸要与31 kDa的催化结构域结合或抑制DNA聚合酶活性，所需量要多10000倍。基于目前的研究结果，讨论了这些长链脂肪酸可能的抑制模式。
