Shuman S
Program in Molecular Biology, Sloan-Kettering Institute, New York, NY 10021.
Proc Natl Acad Sci U S A. 1989 May;86(10):3489-93. doi: 10.1073/pnas.86.10.3489.
Vaccinia virus encapsidates a Mr 32,000 type IDNA topoisomerase. Although the vaccinia gene encoding the topoisomerase is essential for virus growth, the role of the enzyme in vivo remains unclear. In the present study, the physiologic consequences of vaccinia topoisomerase action have been examined in a heterologous system, Escherichia coli. The vaccinia topoisomerase gene was inducibly expressed in an int-lambda lysogen BL21(DE3) using a T7 RNA polymerase-based transcription system. Expression of active topoisomerase in this context resulted in recA-dependent lysogenic induction as well as cell lysis. Surprisingly, topoisomerase expression also effected a 200-fold increase in the titer of infectious lambda phage, apparently by promoting int-independent prophage excision. This effect was not observed during lysogenic induction with nalidixic acid. Restriction analysis of genomic DNA from plaque-purified excisants revealed (in 10 of 10 cases) gross alterations of the DNA structure around the att site relative to the structure of the parental phage DE3. It is construed therefore that vaccinia DNA topoisomerase I acts to promote illegitimate recombination in E. coli.
痘苗病毒包裹着一种分子量为32000的I型DNA拓扑异构酶。尽管编码该拓扑异构酶的痘苗基因对病毒生长至关重要，但该酶在体内的作用仍不清楚。在本研究中，已在异源系统大肠杆菌中研究了痘苗拓扑异构酶作用的生理后果。使用基于T7 RNA聚合酶的转录系统，痘苗拓扑异构酶基因在int-λ溶原菌BL21(DE3)中可诱导表达。在这种情况下，活性拓扑异构酶的表达导致了recA依赖性的溶原诱导以及细胞裂解。令人惊讶的是，拓扑异构酶的表达还使感染性λ噬菌体的滴度增加了200倍，显然是通过促进非int依赖性的原噬菌体切除。在用萘啶酸进行溶原诱导期间未观察到这种效应。对来自噬菌斑纯化的切除产物的基因组DNA进行的限制性分析显示（在10个案例中的10个），相对于亲本噬菌体DE3的结构，att位点周围的DNA结构发生了总体改变。因此可以推断，痘苗DNA拓扑异构酶I在大肠杆菌中起到促进非法重组的作用。
