Inhibition of DNA helicase II unwinding and ATPase activities by DNA-interacting ligands. Kinetics and specificity.
Inhibition of DNA helicase II unwinding and ATPase activities by DNA-interacting ligands. Kinetics and specificity.
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DOI：
10.1016/s0021-9258(19)50072-2
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发表时间：
1992-05
期刊：
影响因子：
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通讯作者：
J. W. George;Sujata V. Ghate;S. W. Matson;J. Besterman
中科院分区：
文献类型：
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作者：
J. W. George;Sujata V. Ghate;S. W. Matson;J. Besterman
尽管DNA解旋酶在DNA加工过程中发挥着重要作用，但人们对DNA相互作用配体对这些解旋酶的影响知之甚少。因此，我们考察了多种DNA结合配体对大肠杆菌DNA解旋酶II催化的解绕和atp酶反应的影响。DNA小凹槽结合剂和简单DNA插入剂对解旋酶II无抑制作用。然而，DNA插入物，如米托蒽醌和诺加霉素，在结合双链DNA的主要凹槽中定位功能，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，利用双链和单链DNA底物的光谱测量了解旋酶II的解绕和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245 bp钝化的完全双链DNA底物，米托蒽醌抑制解绕和atp酶反应的表观Ki值对两种底物都约为1微米，这表明米托蒽醌抑制解旋酶II的机制对两种底物都是相同的，并且需要双链结构的存在。为了加强这一结论，我们采用poly（dT）和热变性后245-bp底物两种单链底物来测定米托蒽醌对解旋酶II的dna依赖性atp酶活性的抑制能力。使用任何一种底物，米托蒽醌抑制解旋酶II的atp酶活性的效果都要差得多。因此，这些结果表明，将米托蒽醌插入双链DNA，并在主槽中放置功能，产生阻碍解旋酶II的复合物，从而抑制ATP水解和解绕活性。此外，我们在这里报道，dna结合配体抑制解旋酶I和IV以及大肠杆菌Rep蛋白的解绕活性，表明对解旋酶II的抑制并非该酶所特有。
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.