Inhibition of the B to Z transition in poly(dGdC).poly(dGdC) by covalent attachment of ethidium: equilibrium studies.
Inhibition of the B to Z transition in poly(dGdC).poly(dGdC) by covalent attachment of ethidium: equilibrium studies.
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通过共价连接乙锭抑制聚（dGdC）.聚（dGdC）中的 B 到 Z 转变：平衡研究。
DOI：
10.1021/bi00109a017
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Chaires,JB
中科院分区：
文献类型：
--
作者：
Gilbert,PL;Graves,DE;Chaires,JB
摘要：研究了单叠氮乙啶（乙啶的光反应类似物）对聚(dGdC) > poly （dGdC）和聚(dGm5dC)-poly-(dGm5dC)共价改性对盐诱导的B - Z跃迁的影响。早期的研究表明，单叠氮乙锭（在没有光线的情况下）与母体溴化乙锭结合DNA的方式相同。单叠氮乙啶-DNA复合物在可见光下光解导致光反应类似物与DNA的共价附着。这种形成共价加合物的能力被用来探测插层不可逆加合物对盐诱导的聚(dGdC)-聚（dGdC）和聚(dGm5dC)*聚-(dGm5dC)多核苷酸的B到Z跃迁的影响。在没有药物的情况下，盐诱导的从B到Z结构的转变以高度合作的方式发生。相反，这种协同性随着共价附着药物浓度的增加而减弱。B到Z过渡的抑制程度被量化为共价附着药物浓度的函数。当poly (dGdC)* poly （dGdC）和poly (dGm5dC)-poly （dGm5dC）的浓度为每4个碱基对一个药物结合时，可以实现这种转变的完全抑制。较低浓度的结合药物对这种转变有部分抑制作用。确定了共价键的插入物对B - Z跃迁的能量学的影响，并证明了加合物在高盐条件下有效地将交替共聚物锁定为右手构象。在过去的十年中，随着左旋DNA和最近的平行链DNA结构的发现，DNA的构象灵活性已经得到了很好的证实（van de Sande etal）。, 1988)。自从Pohl和Jovin（1972）观察到盐诱导poly (dGdC)-poly （dGdC）从右到左Z构象的转变，以及Wang等人（1979）随后确定的晶体结构以来，人们对左撇子DNA的生物学相关性及其在基因调控中的可能作用产生了相当大的兴趣。Z-DNA已被证明具有高度的免疫原性，抗体已被产生并用于检测体内系统中Z-DNA的存在，例如果蝇的多烯染色体（Pohl, 1983； Moeller等人，1982；Thomae等人，1983；Nordheim等人，1981；Lancillotti等人，1985）和Chironomus （Jovinet等人，1983；Arndt-Jovin等人，1983）。1982年，诺德海姆和他的同事从果蝇身上分离出了一类与Z-DNA特异性结合的蛋白质。在类人猿DNA肿瘤病毒（SV40）小染色体的转录增强子区发现了Z-DNA区域（Nordheim et al., 1983），提示Z-DNA可能是一种正调控转录信号。在失活的爪蟾Met-tRNA基因中，逐渐去除具有形成Z-DNA潜力的片段导致转录再激活（Hipskind & Clarkson, 1983），这表明Z-DNA也可能作为降低转录水平的负调控信号。这些发现提示了Z-DNA的自然存在及其在基因表达中的可能作用。然而，确切的作用
Revised Manuscript Received August 26, 1991 abstract: The effects of covalent modification of poly (dGdC> poly (dGdC) and poly (dGm5dC)-poly-(dGm5dC) by ethidium monoazide (a photoreactive analogue of ethidium) on the salt-induced B to Z transition are examined. Earlier studies have shown ethidium monoazide to bind DNA (in the absence of light) in a manner identical to that of the parent ethidium bromide. Photolysis of the ethidium monoazide-DNA complex with visible light results in the covalent attachment of the photoreactive analogue to the DNA. This ability to form a covalent adduct was utilized toprobe the effects of an intercalating irreversibly bound adduct on the salt-induced B to Z transition of the poly (dGdC)-poly (dGdC) and poly (dGm5dC)* poly-(dGm5dC) polynucleotides. In the absence of drug, the salt-induced transition from the B to Z structure occurs in a highly cooperative manner. In contrast, this cooperativity is diminished as the concentration of covalently attached drug is increased. The degree of inhibition of the B to Z transition is quantitated as a function of the concentration of covalently attached drug. At a concentration of one drug bound per four base pairs for poly (dGdC)* poly (dGdC) and seven base pairs for poly (dGm5dC)-poly (dGm5dC), total inhibition of this transition is achieved. Lower concentrations of bound drug were effective in the partial inhibition of this transition. The effects of the covalently bound intercalator on the energetics of the B to Z transition were determined and demonstrated that the adduct is effective in locking the alternating copolymer in a right-handed conformation under high salt conditions. e conformational flexibility of DNA has been well-es-tablished over the last decade with the discoveries of left-handed DNA and most recentlyparallel strand DNA struc-tures (van de Sande etal., 1988). Since the observation of the salt-inducedtransition of poly (dGdC)-poly (dGdC) from a right-to left-handed Z conformationby Pohl and Jovin (1972) and subsequent determination of the crystallographic structure by Wang et al.(1979), considerable interest has been generated concerning the biological relevance of left-handed DNA and its possible roles in gene regulation. Z-DNA has been shown to be highly immunogenic, and antibodies have been produced and used todetect the occurrence of Z-DNA in vivo systems, such as polytene chromo-somes from Drosophila (Pohl, 1983; Moeller et al., 1982; Thomae et al., 1983; Nordheim et al., 1981; Lancillotti et al., 1985) and Chironomus (Jovinet al., 1983; Arndt-Jovin et al., 1983). In 1982, Nordheim and co-workers isolated a class of proteins from Drosophila that bind specifically to Z-DNA. Regions of Z-DNA have been found in the transcriptional enhancer region of the simian DNA tumor virus (SV40) minichromosome (Nordheim et al., 1983), suggesting that Z-DNA may act as a positive regulatory transcription signal. The gradual removal of a segment with the potential of forming Z-DNA in an inactivated Xenopus Met-tRNA gene resulted in transcriptional reactivation (Hipskind & Clarkson, 1983), indicating that Z-DNA may also act as a negative regulatory signal decreasing thelevel of transcription. These findings indicate the natural occurrence of Z-DNA and its possible role in gene expression. However, the precise role of