Blakely G W, Davidson A O, Sherratt D J
Division of Molecular Genetics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
J Biol Chem. 2000 Apr 7;275(14):9930-6. doi: 10.1074/jbc.275.14.9930.
Successful segregation of circular chromosomes in Escherichia coli requires that dimeric replicons, produced by homologous recombination, are converted to monomers prior to cell division. The Xer site-specific recombination system uses two related tyrosine recombinases, XerC and XerD, to catalyze resolution of circular dimers at the chromosomal site, dif. A 33-base pair DNA fragment containing the 28-base pair minimal dif site is sufficient for the recombinases to mediate both inter- and intramolecular site-specific recombination in vivo. We show that Xer-mediated intermolecular recombination in vitro between nicked linear dif "suicide" substrates and supercoiled plasmid DNA containing dif is initiated by XerC. Furthermore, on the appropriate substrate, the nicked Holliday junction intermediate formed by XerC is converted to a linear product by a subsequent single XerD-mediated strand exchange. We also demonstrate that a XerC homologue from Pseudomonas aeruginosa stimulates strand cleavage by XerD on a nicked linear substrate and promotes initiation of strand exchange by XerD in an intermolecular reaction between linear and supercoiled DNA, thereby reversing the normal order of strand exchanges.
大肠杆菌中环状染色体的成功分离要求通过同源重组产生的二聚体质粒在细胞分裂前转化为单体。Xer位点特异性重组系统利用两种相关的酪氨酸重组酶XerC和XerD，在染色体位点dif处催化环状二聚体的拆分。一个包含28个碱基对最小dif位点的33个碱基对DNA片段足以让重组酶在体内介导分子间和分子内的位点特异性重组。我们发现，体外Xer介导的带切口线性dif“自杀”底物与含dif的超螺旋质粒DNA之间的分子间重组由XerC启动。此外，在合适的底物上，由XerC形成的带切口霍利迪连接中间体通过随后单个XerD介导的链交换转化为线性产物。我们还证明，来自铜绿假单胞菌的XerC同源物刺激XerD对带切口线性底物的链切割，并在分子间反应中促进XerD在线性和超螺旋DNA之间的链交换起始，从而颠倒了正常的链交换顺序。
