SITES OF REACTION OF ESCHERICHIA-COLI DNA GYRASE ON PBR322 INVIVO AS REVEALED BY OXOLINIC ACID-INDUCED PLASMID LINEARIZATION
SITES OF REACTION OF ESCHERICHIA-COLI DNA GYRASE ON PBR322 INVIVO AS REVEALED BY OXOLINIC ACID-INDUCED PLASMID LINEARIZATION
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DOI：
10.1016/0022-2836(85)90324-9
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发表时间：
1985-01-01
影响因子：
5.6
通讯作者：
MORRIS, DR
中科院分区：
文献类型：
--
作者：
LOCKSHON, D;MORRIS, DR
pBR 322 DNA，通过溶解oxolinic酸处理的E.用十二烷基硫酸钠纯化大肠杆菌菌株DK 6 recA-（pBR 322），在4种脱氧核苷三磷酸存在下用DNA聚合酶处理，并连接到含有XhoI识别序列的DNA接头上。转化大肠杆菌后产生的耐药菌落大多数为耐药菌落。用这种材料构建的大肠杆菌携带的质粒经限制性酶切分析显示与pBR 322的区别仅在于引入了XhoI位点。来自93个转化体的质粒中的XhoI位点不均匀地分布在pBR 322图谱周围。对这些质粒中的36个进行Maxam-Gilbert DNA序列分析，在XhoI位点的5“末端进行标记，发现其中29个质粒除了XhoI接头外，还含有围绕接头的pBR 322序列的4个碱基对的重复。因此，oxolinic酸诱导的线性化一定导致4个碱基的5“-末端延伸，已知该构型是由oxolinic酸诱导的DNA体外被DNA促旋酶切割而产生的。因此，序列数据允许确定线性化发生的精确点，显然是通过体内存在的回旋酶-DNA共价中间体的流产。当比较29个质粒的19个不同位点时，可以得出以下一组规则：图形 **。其中N是任何核苷酸，R是嘌呤，Y是嘧啶。解理发生在从左起第8和第9位置之间的线处。括号中的G和T分别比T和G更受欢迎;从左起第13位的T和G同样受欢迎。这些规则中的几个与先前基于几个体外促旋酶切割位点提出的规则类似。一些规则显示二分体对称轴之间的分裂点在2股中间，而其他人是明显的不对称。
pBR322 DNA, linearized by lysis of an oxolinic acid-treated culture of E. coli strain DK6recA- (pBR322) with sodium dodecyl sulfate, was purified, treated with DNA polymerase in the presence of the 4 deoxynucleoside triphosphates, and ligated to DNA linkers containing the XhoI recognition sequence. Most of the drug-resistant colonies resulting from transformation of E. coli with this material bore plasmids that appeared by restriction enzyme analysis to differ from pBR322 only by the introduction of an XhoI site. The XhoI sites in plasmids from 93 transformants were distributed unevenly around the pBR322 map. Maxam-Gilbert DNA sequence analysis of 36 of these plasmids, labeled at the 5'' termini of the XhoI sites, revealed that 29 of them contained, in addition to the XhoI linker, a duplication of 4 base-pairs of the pBR322 sequence surrrounding the linker. Therefore, oxolinic acid-induced linearization must have resulted in 5''-terminal extensions of 4 bases, the configuration known to result from oxolinic acid-induced DNA cleavage by DNA gyrase in vitro. The sequence data thus allowed the determination of the precise point at which linearization occurred, apparently by the abortion of a gyrase-DNA covalent intermediate that existed in vivo. When the 19 different sites of the 29 plasmids were compared, the following set of rules could be derived: .**GRAPHIC**. where N is any nucleotide, R is a purine and Y is a pyrimidine. Cleavage occurred at the line between the 8th and 9th positions from the left. The parenthetical G and T were preferred secondarily to T and G, respectively; T and G in the 13th position from the left were equally preferred. Several of these rules are similar to those proposed previously based on several in vitro gyrase cleavage sites. Some of the rules show dyad symmetry around the axis midway between the cleavage points in the 2 strands, while others are distinctly asymmetric.