Preferential DNA repair of (6-4) photoproducts in the dihydrofolate reductase gene of Chinese hamster ovary cells.
Preferential DNA repair of (6-4) photoproducts in the dihydrofolate reductase gene of Chinese hamster ovary cells.
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发表时间：
1989-10
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通讯作者：
D. C. Thomas;D. Okumoto;A. Sancar;V. Bohr
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作者：
D. C. Thomas;D. Okumoto;A. Sancar;V. Bohr
我们已经开发了一种方法来量化基因和基因组内其他特定序列中的（6-4）光产物。该方法利用了以下两种来自大肠杆菌的酶：ABC切酶，一种多功能DNA修复酶，可识别DNA中的许多类型的损伤，以及DNA光解酶，可还原嘧啶二聚体。从紫外光照射的中国仓鼠卵巢细胞中分离DNA，用限制性内切酶消化。在生物紫外线作用下产生的主要光产物嘧啶二聚体，然后通过DNA光解酶处理完全修复。光活化的DNA用ABC切酶处理，在碱性琼脂糖凝胶中电泳，转移到支持膜上，探测特定的基因组序列。ABC切酶在光活化后产生的净切口主要是由于（6-4）光产物的存在。这些加合物是通过测量全长片段在自射线图上的强度降低来定量的。使用这种方法，我们已经证明（6-4）光产物在二氢叶酸还原酶编码序列及其3'侧非编码序列中以相同的频率产生，并且（6-4）光产物的形成在两个序列中都是线性的，直到60 J/m2的紫外线剂量。在4、8和24小时的剂量40 J/m2后，测量了这些DNA序列中（6-4）光产物的修复。在这两个序列中，（6-4）光产物的修复效率都高于嘧啶二聚体，并且与下游非编码序列相比，（6-4）光产物在二氢叶酸还原酶基因中具有优先修复性。
We have developed a method to quantify (6-4) photoproducts in genes and other specific sequences within the genome. This approach utilizes the following two enzymes from Escherichia coli: ABC excinuclease, a versatile DNA repair enzyme which recognizes many types of lesions in DNA, and DNA photolyase, which reverts pyrimidine dimers. DNA is isolated from UV irradiated Chinese hamster ovary cells and digested with a restriction enzyme. Pyrimidine dimers, the major photoproduct produced at biological UV fluences, are then completely repaired by treatment with DNA photolyase. The photoreactivated DNA is treated with ABC excinuclease, electrophoresed in an alkaline agarose gel, transferred to a support membrane and probed for specific genomic sequences. Net incisions produced by ABC excinuclease following photoreactivation are largely due to the presence of (6-4) photoproducts. These adducts are quantitated by measuring the reduction of intensity of the full length fragments on the autoradiogram. Using this approach we have shown that (6-4) photoproducts are produced at equal frequency in the dihydrofolate reductase coding sequence and in its 3'-flanking, noncoding sequences and that the formation of (6-4) photoproducts is linear in both sequences up to a UV dose of 60 J/m2. The repair of (6-4) photoproducts in these DNA sequences was measured after a dose of 40 J/m2 over 4-, 8-, and 24-h time periods. The (6-4) photoproducts are repaired more efficiently than pyrimidine dimers in both sequences and there is preferential repair of (6-4) photoproducts in the dihydrofolate reductase gene compared with the downstream, noncoding sequences.