Zou Y, Van Houten B, Farrell N
Department of Chemistry, Virginia Commonwealth University, Richmond 23284-2006.
Biochemistry. 1994 May 10;33(18):5404-10. doi: 10.1021/bi00184a007.
The sequence specificity of interstrand cross-links induced in DNA by mononuclear and dinuclear platinum complexes in a 49-base-pair DNA duplex has been determined directly. This new assay takes advantage of the fact that 3'-->5' exonuclease digestion of randomly platinated DNA produces a pool of fragments of different lengths. This treatment allows identification of the spectrum of adducts impeding the exonuclease scission. Interstrand cross-linked adducts produce fragments that may remain complementary in the proximity of the binding site. As a result, these fragments may act as primer templates for extension upon subsequent treatment with a DNA polymerase. This extension increases the size of the oligonucleotide fragments, which may be evidenced by a more slowly migrating band on a sequencing gel. Concomitantly, the original band corresponding to the digested cross-link decreases in intensity. Therefore, comparison of a sequencing gel after digestion only and after the "digestion-extension" treatment should show the disappearance, or diminished band intensity, of only those fragments with interstrand cross-links. This approach was applied to the analysis of DNA interstrand cross-links formed by cis-[PtCl2(NH3)2] (cis-DDP) and [(trans-PtCl(NH3)2)2H2N(CH2)4NH2]Cl2. Cis-DDP was confirmed to form interstrand cross-links at d(GC) sequences but, interestingly, interstrand cross-links predominated in a sequence GCGG, with possible 1,3-intrastrand but no 1,2-intrastrand cross-links forming. The dinuclear compound formed 1,2, 1,3, and 1,4 DNA interstrand cross-links between guanines on opposite strands. In 1,3 and 1,4 cross-links, the guanines are separated by one and two base pairs, respectively, whereas a 1,2 cross-link is formed from guanines on neighboring base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)
已直接测定了单核和双核铂配合物在49个碱基对的DNA双链体中诱导形成的链间交联的序列特异性。这种新方法利用了这样一个事实，即对随机铂化的DNA进行3'→5'外切核酸酶消化会产生不同长度的片段池。这种处理使得能够鉴定阻碍外切核酸酶切割的加合物谱。链间交联加合物产生的片段在结合位点附近可能保持互补。因此，这些片段在用DNA聚合酶进行后续处理时可作为引物模板进行延伸。这种延伸增加了寡核苷酸片段的大小，这可通过测序凝胶上迁移较慢的条带得到证明。同时，对应于消化交联的原始条带强度降低。因此，仅消化后和“消化 - 延伸”处理后的测序凝胶比较应显示，只有那些具有链间交联的片段会消失或条带强度减弱。该方法应用于分析顺式 - [PtCl2(NH3)2]（顺铂）和[(反式 - PtCl(NH3)2)2H2N(CH2)4NH2]Cl2形成的DNA链间交联。已证实顺铂在d(GC)序列处形成链间交联，但有趣的是，在序列GCGG中链间交联占主导，可能形成1,3 - 链内交联但未形成1,2 - 链内交联。双核化合物在相反链上的鸟嘌呤之间形成1,2、1,3和1,4 DNA链间交联。在1,3和1,4交联中，鸟嘌呤分别被一个和两个碱基对隔开，而1,2交联由相邻碱基对上相邻的鸟嘌呤形成。（摘要截短于250字）
