Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, WBSB 314, 725 North Wolfe Street, Baltimore, Maryland 21205, United States.
Biochemistry. 2011 Feb 8;50(5):882-90. doi: 10.1021/bi101813h. Epub 2011 Jan 11.
Bifunctional DNA alkylating agents form a diverse assortment of covalent DNA interstrand cross-linked (ICL) structures that are potent cytotoxins. Because it is implausible that cells could possess distinct DNA repair systems for each individual ICL, it is believed that common structural and dynamic features of ICL damage are recognized, rather than specific structural characteristics of each cross-linking agent. Investigation of the structural and dynamic properties of ICLs that might be important for recognition has been complicated by heterogeneous incorporation of these lesions into DNA. To address this problem, we have synthesized and characterized several homogeneous ICL DNAs containing site-specific staggered N4-cytosine-ethyl-N4-cytosine cross-links. Staggered cross-links were introduced in two ways, in a manner that preserves the overall structure of B-form duplex DNA and in a manner that highly distorts the DNA structure, with the goal of understanding how structural and dynamic properties of diverse ICL duplexes might flag these sites for repair. Measurements of base pair opening dynamics in the B-form ICL duplex by (1)H NMR line width or imino proton solvent exchange showed that the guanine base opposite the cross-linked cytosine opened at least 1 order of magnitude more slowly than when in a control matched normal duplex. To a lesser degree, the B-form ICL also induced a decrease in base pair opening dynamics that extended from the site of the cross-link to adjacent base pairs. In contrast, the non-B-form ICL showed extensive conformational dynamics at the site of the cross-link, which extended over the entire DNA sequence. Because DNA duplexes containing the B-form and non-B-form ICL cross-links have both been shown to be incised when incubated in mammalian whole cell extracts, while a matched normal duplex is not, we conclude that intrinsic DNA dynamics is not a requirement for specific damage incision of these ICLs. Instead, we propose a general model in which destabilized ICL duplexes serve to energetically facilitate binding of DNA repair factors that must induce bubbles or other distortions in the duplex. However, the essential requirement for incision is an immobile Y-junction where the repair factors are stably bound at the site of the ICL, and the two DNA strands are unpaired.
双功能 DNA 烷化剂形成了多种多样的共价 DNA 链间交联 (ICL) 结构，这些结构是有效的细胞毒素。因为细胞不太可能拥有针对每种 ICL 的独特 DNA 修复系统，所以人们认为 ICL 损伤的共同结构和动态特征是被识别的，而不是每个交联剂的特定结构特征。对可能对识别很重要的 ICL 的结构和动态特性的研究受到这些损伤在 DNA 中不均匀掺入的阻碍。为了解决这个问题，我们合成并表征了几种含有特异性交错 N4-胞嘧啶-乙基-N4-胞嘧啶交联的均相 ICL DNA。交错交联以两种方式引入，一种方式保持 B 型双螺旋 DNA 的整体结构，另一种方式高度扭曲 DNA 结构，目的是了解不同 ICL 双螺旋的结构和动态特性如何标记这些修复位点。通过 (1)H NMR 线宽或亚氨基质子溶剂交换测量 B 型 ICL 双螺旋中的碱基对打开动力学，发现交联胞嘧啶对面的鸟嘌呤碱基的打开速度至少慢一个数量级，而在对照匹配的正常双螺旋中则不然。在较小程度上，B 型 ICL 也诱导了从交联位点到相邻碱基对的碱基对打开动力学的降低。相比之下，非 B 型 ICL 在交联位点显示出广泛的构象动力学，延伸到整个 DNA 序列。因为含有 B 型和非 B 型 ICL 交联的 DNA 双螺旋在孵育于哺乳动物全细胞提取物中时都被证明会被切割，而匹配的正常双螺旋则不会，所以我们得出结论，内在的 DNA 动力学不是这些 ICL 特异性损伤切割的要求。相反，我们提出了一个一般模型，其中不稳定的 ICL 双螺旋有助于结合 DNA 修复因子，这些因子必须在双螺旋中诱导泡或其他扭曲。然而，切割的基本要求是一个不可移动的 Y 形结，修复因子在 ICL 位点稳定结合，两条 DNA 链未配对。
