Shafferman A, Flashner Y, Hertman I
Department of Biochemistry, Israel Institute for Biological Research, Ness-Ziona.
J Biomol Struct Dyn. 1983 Dec;1(3):729-42. doi: 10.1080/07391102.1983.10507478.
A short DNA palindrom, produced by head to head ligation of a 29 bp DNA fragment, was inserted into a 27,000 bp plasmid DNA element composed of two functional replicons (R6K, ColE1). Several plasmid types containing a single copy of this palindrom in different locations of insertion on the R6K sequence were obtained. The palindrom was engineered to possess a unique EcoRI recognition sequence at its axis of symmetry. The presence of this restriction site allowed to monitor the genetic stability of the artificial palindrom at their different insertion loci. Out of 5 different insertion locations, one (in pAS807) was found to lead to a significant destabilization of the palindrom. This insertion site lies within the replication control region of R6K. We have shown that the inserted palindrom in pAS8O7 does not affect the functionality of the R6K replication origins. Excission of the palindrom sequences from pAS8O7 was not accompanied by loss of the adjacent R6K DNA sequences. Different deletion derivatives of pAS807 were generated in-vitro in order to determine the driving unit of DNA sequences around the palindrom that are involved in its excision. The results imply that large DNA structure(s) around the palindrom are involved in its excission. Complete deletion of R6K sequences from either the left or the right side of the palindrom resulted in new configurations which stabilized the palindrom. A configuration of R6K DNA sequences exceeding 270 bp long sequence from both sides of the palindrom are necessary for the transition from a palindrom stable to palindrom unstable state. In addition evidence is presented to show that the excision process of palindrom sequences requires a functional polymerase I but not the gene product of recA.
将一个29 bp的DNA片段进行头对头连接产生的短DNA回文序列，插入到一个由两个功能性复制子（R6K、ColE1）组成的27,000 bp质粒DNA元件中。获得了几种在R6K序列上不同插入位置含有该回文序列单拷贝的质粒类型。该回文序列经设计在其对称轴处具有一个独特的EcoRI识别序列。这个限制酶切位点的存在使得能够监测人工回文序列在其不同插入位点的遗传稳定性。在5个不同的插入位置中，发现一个位置（在pAS807中）会导致回文序列显著不稳定。这个插入位点位于R6K的复制控制区域内。我们已经表明，pAS8O7中插入的回文序列不影响R6K复制起点的功能。从pAS8O7中切除回文序列时，相邻的R6K DNA序列并未丢失。为了确定参与回文序列切除的其周围DNA序列的驱动单元，在体外产生了pAS807的不同缺失衍生物。结果表明，回文序列周围的大DNA结构参与了其切除过程。从回文序列的左侧或右侧完全删除R6K序列会导致新的构型，从而使回文序列稳定下来。从回文序列两侧超过270 bp长的R6K DNA序列构型对于从回文序列稳定状态转变为不稳定状态是必要的。此外，有证据表明回文序列的切除过程需要功能性的聚合酶I，但不需要recA的基因产物。
