Smith C A, Hanawalt P C
Proc Natl Acad Sci U S A. 1978 Jun;75(6):2598-602. doi: 10.1073/pnas.75.6.2598.
The repair mode of DNA replication has been demonstrated in isolated nuclei from UV-irradiated human cells. Nuclei are incubated in a mixture containing [(3)H]thymidine triphosphate and bromodeoxyuridine triphosphate in a 1:5 ratio. The (3)H at the density of parental DNA in alkaline CsCl density gradients is then a measure of repair. In nuclei prepared from WI38 cells 30 min after irradiation, repair replication is UV dependent and proceeds at approximately the in vivo rate for 5 min. Repair replication is reduced in irradiated nuclei or in nuclei prepared immediately after irradiation. It is Mg(2+)-dependent and stimulated by added ATP and deoxyribonucleoside triphosphates. No repair replication is observed in nuclei from xeroderma pigmentosum (complementation group A) cells. However, upon addition of coliphage T4 endonuclease V, which specifically nicks DNA containing pyrimidine dimers, repair replication is observed in nuclei from irradiated xeroderma pigmentosum cells and is stimulated in WI38 nuclei. The reaction then persists for an hour and is dependent upon added ATP and deoxyribonucleoside triphosphates. The repair label is in stretches of roughly 35 nucleotides, as it is in intact cells. Added pancreatic DNase does not promote UV-dependent repair synthesis. Our results support the view that xeroderma pigmentosum (group A) cells are defective in the incision step of the DNA excision repair pathway, and demonstrate the utility of this system for probing DNA repair mechanisms.
DNA复制的修复模式已在紫外线照射的人类细胞分离细胞核中得到证实。将细胞核在含有[³H]胸苷三磷酸和溴脱氧尿苷三磷酸（比例为1:5）的混合物中孵育。然后，在碱性CsCl密度梯度中，以亲代DNA密度存在的³H可作为修复的指标。在WI38细胞照射后30分钟制备的细胞核中，修复复制依赖于紫外线，且以大约体内速率进行5分钟。在照射后的细胞核或照射后立即制备的细胞核中，修复复制减少。它依赖于Mg²⁺，并受到添加的ATP和脱氧核糖核苷三磷酸的刺激。在着色性干皮病（互补组A）细胞的细胞核中未观察到修复复制。然而，加入噬菌体T4内切核酸酶V（它能特异性切割含有嘧啶二聚体的DNA）后，在照射的着色性干皮病细胞的细胞核中观察到了修复复制，并且在WI38细胞核中受到刺激。该反应随后持续一小时，并依赖于添加的ATP和脱氧核糖核苷三磷酸。修复标记物呈大约35个核苷酸的片段，如同在完整细胞中一样。添加的胰脱氧核糖核酸酶不会促进紫外线依赖的修复合成。我们的结果支持这样的观点，即着色性干皮病（A组）细胞在DNA切除修复途径的切口步骤存在缺陷，并证明了该系统在探究DNA修复机制方面的实用性。
