Brent T P
Biochim Biophys Acta. 1975 Oct 1;407(2):191-9. doi: 10.1016/0005-2787(75)90284-1.
Crude extracts of cultured human lymphoblasts (CCRF-CEM) contain endonuclease activity that cleaves ultraviolet-irradiated DNA in preference to untreated DNA. Purification of this activity was carried out using ultraviolet-irradiated PM2 phage DNA (5000 ergs/mm2) as substrate in the enzyme assay. Since endonuclease specific for depurinated or depyrimidinated DNA might account for the apparent ultraviolet-irradiated DNA-specific activity, fractions derived during purification were also assayed with partially depurinated DNA. Chromatography of a 45-60% (NH4)2SO4 fraction on a Sephadex G-100 column yielded a peak of activity (35 000 daltons) highly active against depurinated DNA but also active for ultraviolet-irradiated DNA. Further purification by DEAE-cellulose chromatography resolved two activities. One was highly specific for depurinated DNA with only minor activity for ultraviolet-irradiated DNA, and was strongly stimulated by Mg2+. The other was non-specifically active against ultraviolet-irradiated or untreated DNA and was independent of Mg2+. Additional studies suggest that neither of these two activities but a third enzyme is responsible for the ultraviolet-irradiated DNA-specific endonuclease activity observed in crude cell extracts.
培养的人淋巴母细胞（CCRF-CEM）的粗提物含有内切核酸酶活性，该活性优先切割紫外线照射的DNA而非未处理的DNA。在酶分析中，以紫外线照射的PM2噬菌体DNA（5000尔格/平方毫米）为底物对该活性进行纯化。由于对脱嘌呤或脱嘧啶DNA具有特异性的内切核酸酶可能解释了明显的紫外线照射DNA特异性活性，因此在纯化过程中得到的各组分也用部分脱嘌呤的DNA进行了分析。在Sephadex G-100柱上对45%-60%硫酸铵组分进行色谱分析，得到一个活性峰（35000道尔顿），该峰对脱嘌呤DNA具有高活性，但对紫外线照射的DNA也有活性。通过DEAE-纤维素色谱进一步纯化得到两种活性。一种对脱嘌呤DNA具有高度特异性，对紫外线照射的DNA只有轻微活性，并且受到Mg2+的强烈刺激。另一种对紫外线照射或未处理的DNA具有非特异性活性，且不依赖于Mg2+。进一步的研究表明，这两种活性都不是导致在粗细胞提取物中观察到的紫外线照射DNA特异性内切核酸酶活性的原因，而是第三种酶起了作用。
