Subia N L, Kogoma T
J Mol Biol. 1986 Jun 5;189(3):389-99. doi: 10.1016/0022-2836(86)90311-6.
rnh mutants harboring pBR322 were found to contain several slowly migrating DNA species when examined by agarose gel electrophoresis. The plasmid DNA from rnh mutants included large molecules, i.e. plasmids two, three or four times the size of a single plasmid unit. That this DNA contained concatemeric plasmid joined in a head-to-tail fashion was determined by digestion with restriction endonucleases that cleaved the monomeric plasmid DNA at a unique site. This treatment resulted in migration of the plasmid DNA at a mobility identical to that of linearized monomeric plasmid by agarose gel electrophoresis. This was confirmed by electron microscopy. Plasmid concatemer formation was detected with several high-copy-number (relaxed type) plasmids but not with low-copy-number (stringent) plasmids. Concatemer formation was dependent on RecA+ and RecF+ functions since several recA and recF mutations abolished concatemer formation. ColE1-type plasmids were previously shown to replicate in rnh mutants in the absence of DNA polymerase I (PolI) activity. This DNA PolI-independent plasmid replication was also examined for its dependence on the recF and recA gene products. rnh- polA(Ts) recF- strains were efficiently transformed with these plasmids at 30 degrees C and 42 degrees C, indicating the presence of DNA PolI-independent replication under recF- conditions. The presence or absence of plasmid replication in rnh- polA- recA(Ts) strains was also examined by measuring the increase in total amounts of plasmid. The results indicated that DNA PolI-independent replication occurred in these triple mutants at 42 degrees C as well as at 30 degrees C. It was concluded that the recombination event giving rise to concatemer formation was not essential for DNA PolI-independent replication in rnh mutants.
当通过琼脂糖凝胶电泳检测时，发现携带pBR322的rnh突变体含有几种迁移缓慢的DNA种类。来自rnh突变体的质粒DNA包括大分子，即单个质粒单位大小的两倍、三倍或四倍的质粒。通过用在单体质粒DNA的独特位点切割的限制性内切酶消化，确定这种DNA包含以头对尾方式连接的串联质粒。这种处理导致质粒DNA在琼脂糖凝胶电泳中的迁移率与线性化单体质粒相同。这通过电子显微镜得到证实。用几种高拷贝数（松弛型）质粒检测到了质粒串联体的形成，但低拷贝数（严谨型）质粒未检测到。串联体的形成依赖于RecA+和RecF+功能，因为几个recA和recF突变消除了串联体的形成。先前已表明，ColE1型质粒在没有DNA聚合酶I（PolI）活性的情况下在rnh突变体中复制。还研究了这种不依赖DNA PolI的质粒复制对recF和recA基因产物的依赖性。rnh- polA(Ts) recF-菌株在30℃和42℃下用这些质粒进行有效转化，表明在recF-条件下存在不依赖DNA PolI的复制。还通过测量质粒总量的增加来检测rnh- polA- recA(Ts)菌株中质粒复制的有无。结果表明，在这些三重突变体中，42℃和30℃时都发生了不依赖DNA PolI的复制。得出的结论是，导致串联体形成的重组事件对于rnh突变体中不依赖DNA PolI的复制不是必需的。
