Hughes K T, Youderian P, Simon M I
Division of Biology, California Institute of Technology, Pasadena 91125.
Genes Dev. 1988 Aug;2(8):937-48. doi: 10.1101/gad.2.8.937.
The bacteriophage P22-based challenge phase selection was used to characterize the binding of Salmonella Hin recombinase to the wild-type hixL and hixR recombination sites, as well as to mutant and synthetic hix sequences in vivo. Hin recombinase binds to the hixL or hixR recombination sites and represses transcription from an upstream promoter in the challenge phage system. Hin-mediated repression results from Hin associating into multimers either prior to binding or during the binding process at the hix operator sites (cooperativity). The ability of Hin multimers to repress transcription is eliminated when the hix 13-bp half-sites are rotated to opposite sides of the DNA helix by inserting 4 bp between them. Insertion of 1 bp between half-sites reduces overall repression. Hin also binds one of the hixL half-sites to repress transcription, but only when high levels of Hin protein are present in the cell. Mutations have been identified in the hix sites that impair Hin binding. Five of the 26 bp in the hix sites are critical; sites with base-pair substitutions at these five positions show greatly reduced binding. Three additional base pairs make minor contributions to binding. These results are consistent with the results of binding studies between Hin and the hix sites in vitro.
基于噬菌体P22的挑战阶段选择被用于在体内表征鼠伤寒沙门氏菌Hin重组酶与野生型hixL和hixR重组位点以及突变和合成hix序列的结合。在挑战噬菌体系统中，Hin重组酶与hixL或hixR重组位点结合，并抑制上游启动子的转录。Hin介导的抑制作用源于Hin在结合之前或在hix操纵位点的结合过程中形成多聚体（协同作用）。当通过在hix的13 bp半位点之间插入4 bp将它们旋转到DNA螺旋的相对侧时，Hin多聚体抑制转录的能力就会丧失。在半位点之间插入1 bp会降低整体抑制作用。Hin也会与hixL的一个半位点结合以抑制转录，但仅在细胞中存在高水平的Hin蛋白时才会发生。已经在hix位点中鉴定出损害Hin结合的突变。hix位点的26个碱基对中有5个是关键的；在这五个位置发生碱基对替换的位点显示出结合能力大大降低。另外三个碱基对在结合中起较小作用。这些结果与体外Hin和hix位点之间的结合研究结果一致。
