Absalon M J, Wu W, Kozarich J W, Stubbe J
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1995 Feb 14;34(6):2076-86. doi: 10.1021/bi00006a030.
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 degree site, however, is probable. The requirement in the ds-cleavage process for O2, 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.
利用发夹寡核苷酸（阿巴隆等人，1995年）以及我们最近开发的用于确定DNA氧化降解时序列特异性同位素效应的技术（科扎里奇等人，1989年；沃思等人，1993年），在特定序列内的特定位点研究了铁-博来霉素介导的DNA双链切割机制。当参与双链断裂的任何一个核苷酸的C-4'氢被氘取代时，观察到了对双链切割的同位素效应。在详细研究的四个位点上，同一位置发生的单链和双链事件的同位素效应值没有差异。结果与双链切割机制一致，即导致单链和双链切割的途径在从参与切割的第一个核苷酸上夺取C-4'氢之后，从一个共同中间体开始分支。在双链断裂的主要切割位点进行氘取代，未能对次要切割位点的切割量产生同等影响，这表明双链切割可能从参与双链切割事件的任何一个核苷酸开始。然而，在1°位点起始切割可能存在动力学偏好。除了形成“活化博来霉素”所需的氧气外，双链切割过程中对氧气的需求已通过在厌氧条件下进行的反应中没有双链切割产物而得到明确证明，在这种条件下单链切割仍会发生。这些结果部分支持了斯特格纳和波维尔克提出的双链切割基本模型[（1990年）《美国国家科学院院刊》87，8350 - 8354]，其中单个博来霉素分子实现双链切割，并且需要重新活化才能在第二条链上进行切割。建立特定位点单链与双链切割比例的关键因素可能与铁-博来霉素在第二个位点重新活化和/或重新定位以进行切割的效率有关。
