Friedman-Ohana R, Cohen A
Department of Molecular Biology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel 91010, USA.
Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6909-14. doi: 10.1073/pnas.95.12.6909.
The formation of heteroduplex joints in Escherichia coli recombination is initiated by invasion of double-stranded DNA by a single-stranded homologue. To determine the polarity of the invasive strand, linear molecules with direct terminal repeats were released by in vivo restriction of infecting chimeric phage DNA and heteroduplex products of intramolecular recombination were analyzed. With this substrate, the invasive strand is expected to be incorporated into the circular crossover product and the complementary strand is expected to be incorporated into the reciprocal linear product. Strands of both polarities were incorporated into heteroduplex structures, but only strands ending 3' at the break were incorporated into circular products. This result indicates that invasion of the 3'-ending strand initiates the heteroduplex joint formation and that the complementary 5'-ending strand is incorporated into heteroduplex structures in the process of reciprocal strand exchange. The polarity of the invasive strand was not affected by recD, recJ, or xonA mutations. However, xonA and recJ mutations increased the proportion of heteroduplexes containing 5'-ending strands. This observation suggests that RecJ exonuclease and exonuclease I may enhance recombination by degrading the displaced strands during branch migration and thereby causing strand exchange to be unidirectional.
在大肠杆菌重组过程中，异源双链体接头的形成是由单链同源物侵入双链DNA引发的。为了确定侵入链的极性，通过体内限制感染性嵌合噬菌体DNA释放具有直接末端重复序列的线性分子，并分析分子内重组的异源双链产物。对于这种底物，预期侵入链会被整合到环状交叉产物中，而互补链会被整合到反向线性产物中。两种极性的链都被整合到异源双链结构中，但只有在断裂处3'端结束的链被整合到环状产物中。这一结果表明，3'端链的侵入启动了异源双链体接头的形成，并且互补的5'端链在反向链交换过程中被整合到异源双链结构中。侵入链的极性不受recD、recJ或xonA突变的影响。然而，xonA和recJ突变增加了含有5'端链的异源双链体的比例。这一观察结果表明，RecJ核酸外切酶和核酸外切酶I可能通过在分支迁移过程中降解被置换的链来增强重组，从而使链交换成为单向的。
