Bockrath R, Li B H
Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis 46202-5120, USA.
Mutat Res. 1997 May 1;383(3):231-42. doi: 10.1016/s0921-8777(97)00006-2.
UV-irradiated excision proficient Escherichia coli were exposed to light for photoenzymatic reversal (PR) of cyclobutane pyrimidine dimers (CPD) and assayed for reversion mutation (glutamine tRNA suppressor mutations) on semi-enriched medium or on the same medium containing acriflavine to inhibit excision repair. The initial mutation frequency without PR was relatively greater when assayed with acriflavine, and this difference increased as larger UV fluences were used. The PR kinetics were first order and about the same or slightly faster when cells were assayed with acriflavine (after 15, 30 or 45 J/m2, respectively). The results indicated mutation targeting by CPD in excision proficient cells. These results and conclusion contrast sharply with the original study of this type done several years ago. PR kinetics were considerably slower with assays containing acriflavine, sustaining the idea that PR causes repair of non-dimer targeting lesions by enhancing excision repair. To explain this contrast we devised a fluence-decrement rate for estimating the effectiveness of PR and measured PR-dependent excision repair (PER) as the difference in the fluence-decrement rate with excision proficient and deficient cells. PER was more evident when cells were prepared as in the original study but was still an insufficient factor. More importantly, the original study included a component of indirect photoreactivation or photoprotection (using unfiltered PR light) which accentuated the role of excision repair. Taking these factors into account, the original data also are consistent with the model that glutamine tRNA suppressor mutations produced by UV-mutagenesis in excision proficient E. coli result from targeting by CPD just as in excision defective cells. Thus, with regards to a common UV mutation assay, there does not appear to be two types of targeting lesion depending on excision proficiency.
将经紫外线照射且切除功能正常的大肠杆菌暴露于光照下，以进行环丁烷嘧啶二聚体（CPD）的光酶促逆转（PR），并在半富集培养基或含有吖啶黄素以抑制切除修复的相同培养基上测定回复突变（谷氨酰胺tRNA抑制突变）。在用吖啶黄素测定时，未经PR处理的初始突变频率相对较高，并且随着紫外线剂量的增加，这种差异也会增大。PR动力学为一级动力学，当用吖啶黄素测定细胞时（分别在15、30或45 J/m2之后），其动力学大致相同或稍快。结果表明，在切除功能正常的细胞中，CPD可导致突变靶向。这些结果和结论与几年前进行的此类原始研究形成了鲜明对比。在含有吖啶黄素的测定中，PR动力学要慢得多，这支持了PR通过增强切除修复来修复非二聚体靶向损伤的观点。为了解释这种差异，我们设计了一种剂量递减率来估计PR的有效性，并将PR依赖的切除修复（PER）测量为切除功能正常和缺陷细胞之间剂量递减率的差异。当按照原始研究制备细胞时，PER更为明显，但仍然是一个不充分的因素。更重要的是，原始研究包括间接光复活或光保护的成分（使用未过滤的PR光），这突出了切除修复的作用。考虑到这些因素，原始数据也与以下模型一致：在切除功能正常的大肠杆菌中，紫外线诱变产生的谷氨酰胺tRNA抑制突变与切除缺陷细胞一样，也是由CPD靶向引起的。因此，对于常见的紫外线突变测定，似乎不存在取决于切除功能的两种类型的靶向损伤。
