Bonura T, Radany E H, McMillan S, Love J D, Schultz R A, Edenberg H J, Friedberg E C
Biochimie. 1982 Aug-Sep;64(8-9):643-54. doi: 10.1016/s0300-9084(82)80104-1.
Pyrimidine dimer (PD)-DNA glycosylase activity has been reported in both the M. luteus and phage T4 UV endonucleases. In the present studies the T4 PD-DNA glycosylase has been purified close to physical homogeneity using an assay that measures the release of free thymine from UV-irradiated poly ([H5] dT):poly (dA), after the photo-reversal of thymine-thymine dimers. The activity has also been demonstrated in vivo following infection of UV-irradiated E. coli uvr- cells with phage T4. Under these conditions the T4 PD-DNA glycosylase accounts quantitatively for all thymine-containing PD excised from [3H] labeled E. coli DNA. In vitro the T4 PD-DNA glycosylase has an associated AP endonuclease activity that incises UV-irradiated DNA 3 to the apyrimidinic sites created by the glycosylase. However, the glycosylase/AP endonuclease reaction mechanism in vitro does not appear to be a concerted one. In addition, a T4 phage with a temperature-sensitive mutation in the denV gene shows wild-type levels of survival at the permissive temperature, despite the fact that in vitro, extracts of E. coli infected with this mutant show no detectable phage-coded AP endonuclease at 28 degrees C. Thus the exact role of the T4 AP endonuclease in the incision of UV-irradiated DNA dimer in vivo is not clear. The ratio of excised non-containing nucleotides to dimer-containing nucleotides following infection of UV-irradiated E. coli with phage T4 denV+ yields a calculated average repair patch size of approximately 7 nucleotides. In contrast, the calculated average patch size in uninfected E. coli is approximately 70 nucleotides. Thus the extent of excision/resynthesis of UV-irradiated DNA may be determined by the specific mode of incision of the DNA at PD. When uninfected E. coli (uvr+) is exposed to UV radiation, a fraction of the excised thymine-containing PD contain photolabile thymine, suggesting the presence of PD-DNA glycosylase in E. coli. The role of this putative activity in the metabolism of UV-irradiated DNA is under investigation.
在藤黄微球菌和噬菌体T4的紫外线内切核酸酶中均已报道有嘧啶二聚体（PD）-DNA糖基化酶活性。在本研究中，利用一种测定方法，在胸腺嘧啶-胸腺嘧啶二聚体光逆转后，测量从紫外线照射的聚（[H5]dT）:聚（dA）中释放游离胸腺嘧啶的量，已将T4 PD-DNA糖基化酶纯化至接近物理纯的状态。在用噬菌体T4感染紫外线照射的大肠杆菌uvr-细胞后，也在体内证实了该活性。在这些条件下，T4 PD-DNA糖基化酶定量地解释了从[3H]标记的大肠杆菌DNA中切除的所有含胸腺嘧啶的PD。在体外，T4 PD-DNA糖基化酶具有相关的AP内切核酸酶活性，该活性可在糖基化酶产生的无嘧啶位点的3'端切割紫外线照射的DNA。然而，体外糖基化酶/AP内切核酸酶反应机制似乎不是协同的。此外，denV基因发生温度敏感突变的T4噬菌体在允许温度下显示出野生型的存活水平，尽管事实上，用这种突变体感染的大肠杆菌提取物在28℃时未检测到噬菌体编码的AP内切核酸酶。因此，T4 AP内切核酸酶在体内切割紫外线照射的DNA二聚体的确切作用尚不清楚。用噬菌体T4 denV+感染紫外线照射的大肠杆菌后，切除的不含核苷酸与含二聚体核苷酸的比例得出计算出的平均修复片段大小约为7个核苷酸。相比之下，未感染的大肠杆菌中计算出的平均片段大小约为70个核苷酸。因此，紫外线照射的DNA的切除/再合成程度可能由DNA在PD处的特定切割方式决定。当未感染的大肠杆菌（uvr+）暴露于紫外线辐射时，一部分切除的含胸腺嘧啶的PD含有光不稳定的胸腺嘧啶，这表明大肠杆菌中存在PD-DNA糖基化酶。这种假定活性在紫外线照射的DNA代谢中的作用正在研究中。
