Braun J E, Wanamarta A H, Westmijze E J, Wientjes N M, Wijker C A, Lafluer M V, Retèl J
Department of Medical Oncology, Faculty of Medicine, Vrije Universiteit, Amsterdam, Netherlands.
Mutat Res. 1997 Jun 9;384(1):45-53. doi: 10.1016/s0921-8777(97)00014-1.
To investigate a possible role of nucleotide excision repair (NER) of E. coli in the removal of gamma-radiation-induced DNA lesions, double-stranded M13mp10 DNA, which contains a part of the lac operon, including the promoter/operator region, the lacZ alpha gene and a 144 basepair (bp) inframe insert in the lacZ alpha gene, as mutational target was gamma-irradiated in a phosphate buffer under N2. Subsequently, the radiation-exposed DNA was transfected to wild-type or NER-deficient (uvrA-) E. coli, mutants in the mutational target selected, followed by characterization of the mutants by sequence analysis. Both the mutations obtained from wild-type and uvrA- E. coli appeared to consist mainly of bp substitutions. However, in contrast to wild-type cells, a relatively large proportion of the mutations obtained from the NER-deficient cells (about 25%) is represented by -1 bp deletions, indicating that NER may be responsible for the removal of lesions which cause this particular type of frameshift. Comparison of the bp substitutions between both E. coli strains showed considerable differences. Thirty per cent of all bp substitutions in the NER-deficient host are T/A-->C/G transitions which are virtually absent in wild-type E. coli. This indicates that NER is involved in the elimination of lesions responsible for these transitions. This may also be true for a part of the lesions which cause C/G-->T/A transitions, which make up 52% of the bp substitutions in uvrA- cells versus 17% in wild-type cells. Strikingly, C/G-->G/C transversions appeared to be only formed in wild-type, where they make up 22% of all bp substitutions, and not in the NER-deficient E. coli. This result suggests, that due to the action of NER, a particular type of mutation may be introduced. A similar indication holds for C/G-->A/T transversions, which are predominant in wild-type (58%) and in the minority in uvrA- cells (15%).
为了研究大肠杆菌核苷酸切除修复（NER）在去除γ射线诱导的DNA损伤中的可能作用，将含有部分乳糖操纵子（包括启动子/操纵子区域、lacZα基因以及lacZα基因中一个144碱基对（bp）的框内插入片段）作为突变靶点的双链M13mp10 DNA在氮气氛围下于磷酸盐缓冲液中进行γ射线照射。随后，将经辐射处理的DNA转染至野生型或NER缺陷型（uvrA-）大肠杆菌中，挑选突变靶点中的突变体，接着通过序列分析对突变体进行表征。从野生型和uvrA-大肠杆菌获得的突变似乎主要由碱基对替换组成。然而，与野生型细胞相比，从NER缺陷型细胞获得的突变中相对较大比例（约25%）表现为-1 bp缺失，这表明NER可能负责去除导致这种特定类型移码的损伤。两种大肠杆菌菌株之间碱基对替换的比较显示出相当大的差异。NER缺陷型宿主中所有碱基对替换的3%是T/A→C/G转换，而在野生型大肠杆菌中几乎不存在。这表明NER参与了对导致这些转换的损伤的消除。对于导致C/G→T/A转换的部分损伤可能也是如此，在uvrA-细胞中此类转换占碱基对替换的52%，而在野生型细胞中为17%。令人惊讶的是，C/G→G/C颠换似乎仅在野生型中形成，占所有碱基对替换的22%，而在NER缺陷型大肠杆菌中未形成。这一结果表明，由于NER的作用，可能会引入一种特定类型的突变。对于C/G→A/T颠换也有类似的迹象，其在野生型中占主导（58%），而在uvrA-细胞中占少数（15%）。
