May 29, 2003

Recombineering with overlapping single-stranded DNA oligonucleotides: testing a recombination intermediate

Proceedings of the National Academy of Sciences of the United States of America
Daiguan YuD L Court

Abstract

A phage lambda-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 ind...Continue Reading

  • References21
  • Citations61
  • References21
  • Citations61

Citations

Mentioned in this Paper

Rec A Recombinases
Alkalescens-Dispar Group
Base Excision Repair
Recombination, Genetic
Genes, Bacterial
Electroporation
Binding (Molecular Function)
Recombinants
Chromosomes
Oligonucleotides

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