Islas A L, Vos J M, Hanawalt P C
Department of Biological Sciences, Stanford University, California 94305-5020.
Cancer Res. 1991 Jun 1;51(11):2867-73.
We have developed a novel procedure to measure interstrand DNA cross-linking in specific DNA sequences. After alkaline denaturation, CsCl gradient equilibrium sedimentation at pH 10.8 is used to resolve cross-linked double-stranded DNA from un-cross-linked single-stranded DNA. The DNA in gradient fractions is slot-blotted and hybridized with 32P-labeled DNA probes for the sequences of interest. After densitometric quantitation of the autoradiograms, the fraction of DNA cross-linked is determined by the ratio of cross-linked DNA to total DNA (the sum of cross-linked and un-cross-linked DNA). We have used this approach to measure the initial levels of production and extent of repair of the photoadducts of 4'-hydroxymethyl-4,5',8-trimethylpsoralen, i.e., both interstrand cross-links and cross-linkable monoadducts, in specific DNA sequences in cultured human cells. Under conditions in which DNA fragments carrying the expressed dihydrofolate reductase gene were extensively modified, with approximately 92% of the fragments cross-linked, only 37% of the fragments containing the unexpressed fms protooncogene were cross-linked. The overall level of cross-linking for bulk DNA was 74%. Within 24 h, 90% of the cross-linking had been removed from the dihydrofolate reductase gene, whereas little removal was detected in fms, and the bulk DNA showed 31% removal. From this study, we conclude that both the introduction and removal of 4'-hydroxymethyl-4,5',8-trimethylpsoralen adducts are dependent upon the target DNA sequence and its transcriptional activity. The implications for DNA repair of chromatin structure and active transcription are discussed in relation to our results.
我们开发了一种新方法来测量特定DNA序列中的链间DNA交联。碱变性后，在pH 10.8条件下进行CsCl梯度平衡沉降，以从未交联的单链DNA中分离出交联的双链DNA。梯度组分中的DNA进行狭槽印迹，并与针对感兴趣序列的32P标记DNA探针杂交。对放射自显影片进行光密度定量后，通过交联DNA与总DNA（交联和未交联DNA之和）的比率来确定DNA交联的比例。我们已使用这种方法来测量培养的人细胞中特定DNA序列中4'-羟甲基-4,5',8-三甲基补骨脂素光加合物的初始产生水平和修复程度，即链间交联和可交联单加合物。在携带表达的二氢叶酸还原酶基因的DNA片段被广泛修饰的条件下，约92%的片段发生交联，而含有未表达的fms原癌基因的片段只有37%发生交联。总体DNA的交联水平为74%。在24小时内，二氢叶酸还原酶基因中90%的交联已被去除，而在fms基因中几乎未检测到去除，总体DNA显示有31%的去除。从这项研究中，我们得出结论：4'-羟甲基-4,5',8-三甲基补骨脂素加合物的引入和去除均取决于目标DNA序列及其转录活性。结合我们的结果讨论了染色质结构和活性转录对DNA修复的影响。
