QUANTITATIVE MEASUREMENT OF SINGLE-STRAND AND DOUBLE-STRAND BREAKAGE OF DNA IN ESCHERICHIA-COLI BY THE ANTITUMOR ANTIBIOTICS BLEOMYCIN AND TALISOMYCIN
QUANTITATIVE MEASUREMENT OF SINGLE-STRAND AND DOUBLE-STRAND BREAKAGE OF DNA IN ESCHERICHIA-COLI BY THE ANTITUMOR ANTIBIOTICS BLEOMYCIN AND TALISOMYCIN
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DOI：
10.1128/aac.27.4.460
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发表时间：
1985-01-01
影响因子：
4.9
通讯作者：
CROOKE, ST
中科院分区：
文献类型：
--
作者：
MIRABELLI, CK;HUANG, CH;CROOKE, ST
开发了一种测定法，其中化学试剂对细胞内DNA的单链断裂（ssb）和双链断裂（dsb）可以准确地定量和区分。E.将含有质粒pBR 322 DNA的大肠杆菌细胞与抗肿瘤抗生素博莱霉素A2（BLM A2）或泰利霉素A（TLM A）一起孵育。分离质粒DNA，然后在1%琼脂糖凝胶上进行电泳分析，以分离以下质粒DNA的构象形式：天然的、共价闭合的、超螺旋的、I型;有切口的、松弛的环状的、II型;和双链断裂的、线性的、III型。通过凝胶的光密度分析进行的定量表明，BLM A2和TLM A在减少细胞中等量的I型DNA所需的药物浓度方面具有相同的活性，而在体外（使用分离的pBR 322 DNA作为药物底物），产生等量的I型DNA减少需要比BLM A2多2倍的TLM A。TLM A比BLM A2产生更多的细胞内dsb。从BLM A2和TLM A测量的细胞内dsb活性（dsb/ssb比率）与当分离的pBR 322 DNA用作底物时对相应试剂测量的那些相等。在大肠coli、ssb和dsb均能修复，但TLM A损伤修复较慢，修复程度较低，这可能反映了dsb的相对频率。
An assay was developed in which single-strand breakage (ssb) and double-strand breakage (dsb) of intracellular DNA by chemical agents can be accurately quantitated and differentiated. E. coli cells containing plasmid pBR322 DNA were incubated with the antitumor antibiotics bleomycin A2 (BLM A2) or talisomycin A (TLM A). The plasmid DNA was isolated and then analyzed by electrophoresis on 1% agarose gels to separate the following conformational forms of plasmid DNA: native, covalently closed, super helical, form I; nicked, relaxed circular, form II; and double-strand broken, linear, form III. Quantitation by densitometric analysis of the gels showed that BLM A2 and TLM A were equally active in terms of the concentrations of drug necessary to reduce equivalent amounts of form I DNA in the cells, whereas in vitro (using isolated pBR322 DNA as a drug substrate) 2-fold more TLM A than BLM A2 was required to produce an equivalent amount of reduction in form I DNA. TLM A produced more intracellular dsb than did BLM A2. The intracellular dsb activities (dsb/ssb ratio) measured from BLM A2 and TLM A were equivalent to those measured for the respective agents when isolated pBR322 DNA was used as the substrate. In E. coli, ssb and dsb were repaired, but TLM A damage was repaired more slowly and to a lesser extent, which may reflect the relative frequency of dsb.