(+)-CC-1065 as a structural probe of Mu transposase-induced bending of DNA: overcoming limitations of hydroxyl-radical footprinting.
(+)-CC-1065 as a structural probe of Mu transposase-induced bending of DNA: overcoming limitations of hydroxyl-radical footprinting.
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( )-CC-1065 作为 Mu 转座酶诱导的 DNA 弯曲的结构探针：克服羟基自由基足迹的局限性。
DOI：
10.1093/nar/21.18.4281
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发表时间：
1993
影响因子：
14.9
通讯作者：
Hurley,LH
中科院分区：
文献类型：
--
作者：
Ding,ZM;Harshey,RM;Hurley,LH
噬菌体Mu转座酶（A蛋白）主要负责Mu基因组的转座。该蛋白质结合到六个att位点上，Mu DNA的每一端各有三个。在大多数att位点，蛋白质单体与DNA的相互作用发生在三个小沟和两个连续的大沟上，并导致高达约90°的弯曲。为了探测这些A蛋白诱导的弯曲的方向和位点，我们使用抗肿瘤抗生素（+）-CC-1065作为结构探针。由于结合在小沟内，（+）-CC-1065能够以序列选择性方式烷基化腺嘌呤的N3。这种选择性部分取决于DNA序列的构象柔性，并且共价加合物具有弯曲的DNA结构，其中发生小沟变窄。在用凝胶阻滞或DNA链断裂来监测A-蛋白- DNA复合物或DNA上（+）-CC-1065烷基化位点（L3后位点）的稳定性的实验中，我们用这种药物证明了与A-蛋白相互作用的三个小沟中，在蛋白结合后，外围的两个小沟是“开放的”或可接近药物键合的。这些药物结合位点很可能代表至少两个A-蛋白诱导的弯曲位点的结合。值得注意的是，在这些位点的弯曲位点间隔约两个螺旋圈，弯曲被认为是通过缩小DNA的小沟而发生的。这两个外周位点之间的中间小沟受到保护，不受（+）-CC-1065烷基化的影响。结果进行了讨论，在参考提出的模型中的A-蛋白质attL 3网站复杂的整体DNA弯曲。这项研究说明了（+）-CC- 1065作为探针的蛋白质诱导的DNA弯曲，以及小沟DNA弯曲蛋白与DNA的相互作用，这可能是在羟基自由基足迹实验掩盖的效用。
Phage Mu transposase (A-protein) Is primarily responsible for transposition of the Mu genome. The protein binds to six att sites, three at each end of Mu DNA. At most att sites Interaction of a protein monomer with DNA Is seen to occur over three minor and two consecutive major grooves and to result in bending up to about 90°. To probe the directionality and locus of these A-protein-lnduced bends, we have used the, antitumor antibiotic (+ )-CC-1065 as a structural probe. As a consequence of binding within the minor groove, ( + )-CC-1065 is able to alky late N3 of adenine in a sequence selective manner. This selectivity Is partially determined by conformatlonal flexibility of the DNA sequence, and the covalent adduct has a bent DNA structure In which narrowing of the minor groove has occurred. Using this drug in experiments in which either gel retardation or DNA strand breakage are used to monitor the stability of the A-proteln - DNA complex or the (+ )-CC-1065 alkylatlon sites on DNA (aft site L3), we have demonstrated that of the three minor grooves implicated in the Interaction with A-proteln, the peripheral two are 'open' or accessible to drug bonding following protein binding. These drug-bonding sites very likely represent binding at at least two A-protelninduced bending sites. Significantly, the locus of bending at these sites is spaced approximately two helical turns apart, and the bending is proposed to occur by narrowing of the minor groove of DNA. The Intervening minor groove between these two peripheral sites is protected from ( + )-CC-1065 alkylatlon. The results are discussed In reference to a proposed model for overall DNA bending in the A-protein att L3 site complex. This study illustrates the utility of ( + )-CC- 1065 as a probe for protein-induced bending of DNA, as well as for interactions of minor groove DNA bending proteins with DNA which may be masked in hydroxyl radical footprinting experiments.