Lopez B S, Corteggiani E, Bertrand-Mercat P, Coppey J
Institut CURIE, section de Biologie, Paris, France.
Nucleic Acids Res. 1992 Feb 11;20(3):501-6. doi: 10.1093/nar/20.3.501.
The involvement of a double strand break in the initiation of homologous recombination was examined in human nuclear extracts. M13 duplex derivatives, containing inserts in the LacZ' region (producing white plaques), were cleaved by restriction enzymes and coincubated in the extracts with a circular plasmid containing the LacZ' region without insert, and unable to produce plaques. Repair was estimated by the ability to produce plaques after transfection into JM109 (recA1) bacteria. Recombination with the plasmid enhances the number of plaques and also the frequency of M13 producing blue plaques. Heterologous insertions in the region surrounding the break were analyzed for their effects on initiation of recombination. The extent of repair by recombination (number of plaques) was compared with the number of blue plaques among the repaired population. Initiation of recombination is inhibited when heterologous insertions are located at 7bp from the break, on the right side as well as on the left side. A low level of recombination is measurable for 27 bp of homology but the maximum efficiency of recombination occurred with homologies of 165 or 320 bp from the break to the heterologous insertion. At 320 bp, the extent of recombinational repair remained at a plateau level but the frequency of blue plaques progressively decreases. We have also analyzed the effect of different sizes of inserts. With longer inserts, a longer length of homology adjacent to the break is required for optimum recombination. However, the size of the insert does not affect the low level of recombination that occurred with a short homology (27 bp). The results indicate that the process is initiated at or near the break, requires homology on both sides of the break and is followed by an elongation from the double strand break to the distal regions of the DNA. Our data provide some support to the double-strand-break repair model established for meiotic recombination in yeast.
在人核提取物中研究了双链断裂在同源重组起始过程中的作用。含有LacZ'区域插入片段（产生白色噬菌斑）的M13双链衍生物被限制酶切割，并在提取物中与不含插入片段且无法产生噬菌斑的含有LacZ'区域的环状质粒共同孵育。通过转染到JM109（recA1）细菌后产生噬菌斑的能力来评估修复情况。与质粒的重组增加了噬菌斑的数量以及产生蓝色噬菌斑的M13的频率。分析了断裂周围区域的异源插入对重组起始的影响。将重组修复的程度（噬菌斑数量）与修复群体中蓝色噬菌斑的数量进行比较。当异源插入位于断裂右侧和左侧7个碱基对处时，重组起始受到抑制。对于27个碱基对的同源性可检测到低水平的重组，但从断裂到异源插入的同源性为165或320个碱基对时，重组效率最高。在320个碱基对时，重组修复的程度保持在平台水平，但蓝色噬菌斑的频率逐渐降低。我们还分析了不同大小插入片段的影响。对于较长的插入片段，断裂附近需要更长的同源长度才能实现最佳重组。然而，插入片段的大小并不影响短同源性（27个碱基对）时发生的低水平重组。结果表明，该过程在断裂处或其附近起始，需要断裂两侧的同源性，随后从双链断裂延伸到DNA的远端区域。我们的数据为酵母减数分裂重组中建立的双链断裂修复模型提供了一些支持。
