George J W, Ghate S, Matson S W, Besterman J M
Department of Biology, University of North Carolina, Chapel Hill 27599.
J Biol Chem. 1992 May 25;267(15):10683-9.
Although DNA helicases play important roles in the processing of DNA, little is known about the effects of DNA-interacting ligands on these helicases. Therefore, the effects of a wide variety of DNA-binding ligands on the unwinding and ATPase reactions catalyzed by Escherichia coli DNA helicase II were examined. DNA minor groove binders and simple DNA intercalators did not inhibit helicase II. However, DNA intercalators, such as mitoxantrone and nogalamycin, which position functionalities in the major groove upon binding duplex DNA, were potent inhibitors of helicase II. To determine the mechanism by which mitoxantrone inhibited helicase II, the unwinding and DNA-dependent ATPase activities of helicase II were measured using a spectrum of double- and single-stranded DNA substrates. Using either a 71-base pair (bp) M13mp7 partially duplexed DNA substrate or a 245-bp bluntended, fully duplexed DNA substrate, the apparent Ki value for inhibition by mitoxantrone of both the unwinding and ATPase reactions was approximately 1 microM for both substrates, suggesting that the mechanism of inhibition of helicase II by mitoxantrone is the same for both substrates and requires the presence of double-stranded structure. To strengthen this conclusion, the ability of mitoxantrone to inhibit the DNA-dependent ATPase activity of helicase II was determined using two single-stranded substrates, poly(dT) and the 245-bp substrate after heat denaturation. Using either substrate, mitoxantrone inhibited the ATPase activity of helicase II far less effectively. Thus, these results indicate that the intercalation of mitoxantrone into double-stranded DNA, with accompanying placement of functionalities in the major groove, generates a complex that impedes helicase II, resulting in both inhibition of ATP hydrolysis and unwinding activity. Furthermore, we report here that DNA-binding ligands inhibit the unwinding activity of helicases I and IV and Rep protein from E. coli, demonstrating that the inhibition observed for helicase II is not unique to this enzyme.
尽管DNA解旋酶在DNA加工过程中发挥着重要作用，但关于与DNA相互作用的配体对这些解旋酶的影响却知之甚少。因此，研究了多种DNA结合配体对大肠杆菌DNA解旋酶II催化的解旋和ATP酶反应的影响。DNA小沟结合剂和简单的DNA嵌入剂不会抑制解旋酶II。然而，DNA嵌入剂，如米托蒽醌和诺加霉素，它们在结合双链DNA时将功能基团定位在大沟中，是解旋酶II的有效抑制剂。为了确定米托蒽醌抑制解旋酶II的机制，使用一系列双链和单链DNA底物测量了解旋酶II的解旋和DNA依赖性ATP酶活性。使用71碱基对（bp）的M13mp7部分双链DNA底物或245bp平端、完全双链DNA底物，米托蒽醌对解旋和ATP酶反应抑制的表观Ki值对于两种底物均约为1μM，这表明米托蒽醌对解旋酶II的抑制机制对于两种底物是相同的，并且需要双链结构的存在。为了强化这一结论，使用两种单链底物，即聚（dT）和热变性后的245bp底物，测定了米托蒽醌抑制解旋酶II的DNA依赖性ATP酶活性的能力。使用任何一种底物时，米托蒽醌对解旋酶II的ATP酶活性的抑制效果都要差得多。因此，这些结果表明，米托蒽醌嵌入双链DNA中，并伴随在大沟中定位功能基团，产生了一种阻碍解旋酶II的复合物，导致ATP水解和解旋活性均受到抑制。此外，我们在此报告，DNA结合配体抑制大肠杆菌解旋酶I和解旋酶IV以及Rep蛋白的解旋活性，表明观察到的对解旋酶II的抑制并非该酶所特有。
