SEQUENCE-SPECIFIC DOUBLE-STRAND CLEAVAGE OF DNA BY FE BLEOMYCIN .2. MECHANISM AND DYNAMICS
SEQUENCE-SPECIFIC DOUBLE-STRAND CLEAVAGE OF DNA BY FE BLEOMYCIN .2. MECHANISM AND DYNAMICS
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DOI：
10.1021/bi00006a030
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发表时间：
1995-02-14
期刊：
影响因子：
2.9
通讯作者：
STUBBE, J
中科院分区：
文献类型：
--
作者：
ABSALON, MJ;WU, W;STUBBE, J
已经使用发夹寡核苷酸在特定序列内的特定位点研究了铁-博来霉素介导的DNA的顺式切割的机制（Absalon等人，1995）和我们最近开发的用于确定DNA氧化降解后的序列特异性同位素效应的技术（Kozarich等人，1989; Worth等人，1993年）。当参与顺式断裂的任一核苷酸的C-4'氢被氘取代时，已经观察到对顺式断裂的同位素效应。在详细检查的四个地点，在同一地点发生的ss和ds事件的同位素效应值是无法区分的。结果与顺式切割机制一致，其中在从参与切割的第一个核苷酸中提取C-4'氢之后，导致顺式和顺式切割的途径从共同中间体中分离出来。氘取代在一级切割位点的DS-断裂未能导致在二级切割位点上的切割量的等效效果，这表明DS-切割可以从参与DS-切割事件的核苷酸中的任一个开始。然而，在1度位点开始裂解的动力学偏好是可能的。除了形成“活化的BLM”所需的外，在顺式裂解过程中对O-2的需要已经通过在厌氧条件下进行的反应中不存在顺式裂解产物而清楚地证明，其中在厌氧条件下仍然发生顺式裂解。这些结果部分支持Steighner和Povirk [（1990）Proc. Natl. Acad. Sci. U. S. A. 87，8350-8354]，其中单个BLM分子影响顺式切割，并且需要重新激活才能影响第二链的切割。在特定位点建立顺式与顺式切割比率的基本因素可能与Fe-BLM可以被重新激活和/或重新定位在第二位点进行切割的效率有关。
The mechanism of iron-bleomycin-mediated ds-cleavage of DNA has been investigated at specific sites within specific sequences using hairpin oligonucleotides (Absalon et al., 1995) and our recently developed technique for determining sequence-specific isotope effects upon oxidative degradation of DNA (Kozarich et al., 1989; Worth et al., 1993). Isotope effects upon ds-cleavage have been observed when the C-4' hydrogen of either nucleotide involved in the ds-break was substituted with deuterium. The values of the isotope effects determined for ss and ds events occurring at the same site were indistinguishable at four sites examined in detail. The results are consistent with a mechanism of ds-cleavage in which the pathways leading to ss- and ds-cleavage partition from a common intermediate subsequent to abstraction of the C-4' hydrogen from the first nucleotide involved in the cleavage. Deuterium substitution at the primary cleavage site of a ds-break failed to result in an equivalent effect on the amount of cleavage at the secondary cleavage site, suggesting that ds-cleavage may be initiated from either of the nucleotides involved in the ds-cleavage event. A kinetic preference for cleavage initiated at the 1 degrees site, however, is probable. The requirement in the ds-cleavage process for O-2, in addition to that needed to form ''activated BLM'', has been clearly demonstrated by the absence of ds-cleavage products in reactions performed under anaerobic conditions in which ss-cleavage still occurs. These results support, in part, the basic model for ds-cleavage proposed by Steighner and Povirk [(1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8350-8354], in which a single molecule of BLM effects ds-cleavage and requires reactivation to effect cleavage at the second strand. The essential factor establishing the ratio of ss- to ds-cleavage at a specific site may be related to the efficiency by which Fe-BLM can be reactivated and/or repositioned at a second site for cleavage.