Luo Y, Gao Q, Deonier R C
Department of Biological Sciences, University of Southern California, Los Angeles 90089-1340, USA.
Mol Microbiol. 1995 Mar;15(5):829-37. doi: 10.1111/j.1365-2958.1995.tb02353.x.
The extent of the F plasmid oriT nicking region was determined from the properties of successive substitution mutations in the region from base pair 121 to base pair 174 and from KMnO4 probing of DNA structural distortions induced in vivo by tra gene products. Nicking and transfer assays indicated that the left margin of oriT lies predominantly at the nick site, and that the nicking domain primarily lies within 17 bp to the right of the nick. Some mutants that were proficient for nicking showed reduced frequencies of termination, indicating that oriT nicking does not guarantee efficient termination. DNA in the vicinity of the nick (G137, T138, G140, and T141 on the nicked strand) showed elevated sensitivity to KMnO4 when tra gene products were present in the donor. Bases C145, C146, C147, C149, and G150 on the unnicked strand also became more sensitive to oxidation under tra+ conditions. The bases preferentially oxidized by KMnO4 lie within the nicking domain, as defined by the substitution mutants, and they include dinucleotides that can produce kinks in the DNA. Base pairs in the nicking region are calculated to be more thermodynamically stable than base pairs in the flanking regions.
通过对121至174碱基对区域内连续取代突变的特性以及对tra基因产物在体内诱导的DNA结构畸变进行高锰酸钾探测，确定了F质粒oriT切口区域的范围。切口和转移分析表明，oriT的左边界主要位于切口位点，且切口结构域主要位于切口右侧17个碱基对范围内。一些具有切口能力的突变体显示出终止频率降低，这表明oriT切口并不能保证有效终止。当供体中存在tra基因产物时，切口附近的DNA（切口链上的G137、T138、G140和T141）对高锰酸钾的敏感性升高。在tra+条件下，未切口链上的碱基C145、C146、C147、C149和G150对氧化也变得更敏感。高锰酸钾优先氧化的碱基位于由取代突变体定义的切口结构域内，并且包括可在DNA中产生扭结的二核苷酸。计算得出，切口区域内的碱基对比侧翼区域内的碱基对在热力学上更稳定。
