Bichara Marc, Pinet Isabelle, Origas Mélanie, Fuchs Robert P P
Departement Intégrité du Génome, UMR 7100, CNRS, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sebastien Brant, BP 10413, 67412 Illkirch-Cedex, France.
DNA Repair (Amst). 2006 Jan 5;5(1):129-37. doi: 10.1016/j.dnarep.2005.08.019. Epub 2005 Oct 28.
Lesions that transiently block DNA synthesis generate replication intermediates with recombinogenic potential. In order to investigate the mechanisms involved in lesion-induced recombination, we developed an homologous recombination assay involving the transfer of genetic information from a plasmid donor molecule to the Escherichia coli chromosome. The replication blocking lesion used in the present assay is formed by covalent binding of the carcinogen N-2-acetylaminofluorene to the C8 position of guanine residues (G-AAF adducts). The frequency of recombination events was monitored as a function of the number of lesions present on the donor plasmid. These DNA adducts are found to trigger high levels of homologous recombination events in a dose-dependent manner. Formation of recombinants is entirely RecA-dependent, the RecF and RecBCD sub-pathways accounting for about 2/3 and 1/3, respectively. Inactivation of recG stimulates recombinant formation about five-fold. In a recG background, the RecF pathway is stimulated about four-fold, while the contribution of the RecBCD pathway remains constant. In addition, in the recG strain, a recombination pathway that accounts for about 30% of the recombinants and requires genes that belong to both RecF and RecBCD pathways is revealed.
短暂阻断DNA合成的损伤会产生具有重组潜能的复制中间体。为了研究损伤诱导重组所涉及的机制，我们开发了一种同源重组检测方法，该方法涉及将遗传信息从质粒供体分子转移到大肠杆菌染色体上。本检测中使用的复制阻断损伤是由致癌物N-2-乙酰氨基芴与鸟嘌呤残基的C8位共价结合形成的（G-AAF加合物）。重组事件的频率作为供体质粒上损伤数量的函数进行监测。发现这些DNA加合物以剂量依赖的方式引发高水平的同源重组事件。重组体的形成完全依赖于RecA，RecF和RecBCD子途径分别占约2/3和1/3。recG的失活刺激重组体形成约五倍。在recG背景下，RecF途径被刺激约四倍，而RecBCD途径的贡献保持不变。此外，在recG菌株中，发现了一种重组途径，该途径约占重组体的30%，并且需要属于RecF和RecBCD途径的基因。
