Wang G, Rahman M S, Humayun M Z
Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, 185 South Orange Avenue, MSB F607, Newark, New Jersey 07103-2714, USA.
Biochemistry. 1997 Aug 5;36(31):9486-92. doi: 10.1021/bi970650o.
In order to characterize mutagenic translesion DNA synthesis in UVM-induced Escherichia coli, we have developed a high-resolution DNA replication system based on E. coli cell extracts and M13 genomic DNA templates bearing mutagenic lesions. The assay is based on the conversion of M13 viral single-stranded DNA (ssDNA) bearing a single site-specific DNA lesion to the double-stranded replicative form (RF) DNA, and permits one to quantitatively measure the efficiency of translesion synthesis. Our data indicate that DNA replication is most strongly inhibited by an abasic site, a classic SOS-dependent noninstructive lesion. In contrast, the efficiency of translesion synthesis across SOS-independent lesions such as O6-methylguanine and DNA uracil is around 90%, very close to the values obtained for control DNA templates. The efficiency of translesion synthesis across 3,N4-ethenocytosine and 1, N6-ethenoadenine is around 20%, a value that is similar to the in vivo efficiency deduced from the effect of the lesions on the survival of transfected M13 ssDNA. Neither DNA polymerase I nor polymerase II appears to be required for the observed translesion DNA synthesis because essentially similar results are obtained with extracts from polA- or polB-defective cells. The close parallels in the efficiency of translesion DNA synthesis in vitro and in vivo for the five site-specific lesions included in this study suggest that the assay may be suitable for modeling mutagenesis in an accessible in vitro environment.
为了表征紫外线诱变剂（UVM）诱导的大肠杆菌中的诱变跨损伤DNA合成，我们基于大肠杆菌细胞提取物和携带诱变损伤的M13基因组DNA模板，开发了一种高分辨率DNA复制系统。该测定基于将带有单个位点特异性DNA损伤的M13病毒单链DNA（ssDNA）转化为双链复制形式（RF）DNA，并允许定量测量跨损伤合成的效率。我们的数据表明，无碱基位点（一种典型的SOS依赖性非指令性损伤）对DNA复制的抑制作用最强。相比之下，跨SOS非依赖性损伤（如O6-甲基鸟嘌呤和DNA尿嘧啶）的跨损伤合成效率约为90%，非常接近对照DNA模板获得的值。跨3,N4-乙撑胞嘧啶和1,N6-乙撑腺嘌呤的跨损伤合成效率约为20%，这一数值与从损伤对转染的M13 ssDNA存活的影响推断出的体内效率相似。观察到的跨损伤DNA合成似乎既不需要DNA聚合酶I也不需要聚合酶II，因为用polA或polB缺陷细胞的提取物获得了基本相似的结果。本研究中包括的五个位点特异性损伤在体外和体内跨损伤DNA合成效率上的密切相似性表明，该测定可能适用于在可及的体外环境中模拟诱变。
