Recombineering with overlapping single-stranded DNA oligonucleotides: Testing a recombination intermediate
Recombineering with overlapping single-stranded DNA oligonucleotides: Testing a recombination intermediate
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DOI：
10.1073/pnas.1232375100
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发表时间：
2003-06-10
影响因子：
11.1
通讯作者：
Court, DL
中科院分区：
文献类型：
--
作者：
Yu, DG;Sawitzke, JA;Court, DL
一种基于噬菌体A的重组系统Red可用于在体内对染色体或表观DNA进行高效的突变、修复和工程。当与其目标具有短侧向同源性的长线性双链DNA用于重组时，需要lambda Exo、Beta和Gam蛋白。目前的模型是：(I)GAM抑制宿主RecBCD的活性，从而保护DNA底物进行重组；(Ii)Exo从DNA的每个末端沿5‘-gt；3’方向降解，产生带有3‘-单链DNA尾巴的双链DNA；(Iii)Beta结合这些3’端突出物，以保护它们并使其成为互补序列。我们已经测试了这个模型的Red重组，使用电穿孔引入重叠的，互补的寡核苷酸，当在体内退火时，近似于Exo应该产生的重组中间产物。利用这一技术，我们发现了外源无关的重组。令人惊讶的是，类似结构的5‘端悬挑底物的重组效率更高。这种5‘端的悬垂重组需要Exo和Beta两者才能进行高水平的重组，并且这两个寡核苷酸在其3’端只需要重叠6bp。结果表明，Exo可以将Beta装载到其产生的3‘端突出物上。此外，多个重叠的寡核苷酸被成功地用于在体内产生重组体，这一技术可能被证明对许多基因工程程序有用。
A phage A-based recombination system, Red, can be used for high-efficiency mutagenesis, repair, and engineering of chromosomal or episomal DNA in vivo in Escherichia coli. When long linear double-stranded DNA with short flanking homologies to their targets are used for the recombination, the lambda Exo, Beta, and Gam proteins are required. The current model is: (i) Gam inhibits the host RecBCD activity, thereby protecting the DNA substrate for recombination; (ii) Exo degrades from each DNA end in a 5'-->3' direction, creating double-stranded DNA with 3' single-stranded DNA tails; and (iii) Beta binds these 3' overhangs to protect and anneal them to complementary sequences. We have tested this model for Red recombination by using electroporation to introduce overlapping, complementary oligonucleotides that when annealed in vivo approximate the recombination intermediate that Exo should create. Using this technique we found Exo-independent recombination. Surprisingly, a similarly constructed substrate with 5' overhangs recombined more efficiently. This 5' overhang recombination required both Exo and Beta for high levels of recombination and the two oligonucleotides need to overlap by only 6 bp on their 3' ends. Results indicate that Exo may load Beta onto the 3' overhang it produces. In addition, multiple overlapping oligonucleotides were successfully used to generate recombinants in vivo, a technique that could prove useful for many genetic engineering procedures.