Recombination with single-strand DNA oligonucleotides (oligos) in E. coli is an efficient and rapid way to modify replicons in vivo. The generation of a nucleotide alteration by oligo recombination provides novel assays for studying cellular processes. Single-strand exonucleases inhibit oligo recombination, and by mutating all four known exonucleases recombination is increased. Increasing the oligo concentration or addition of non-specific carrier oligo titrates out the exonucleases. In a model for oligo recombination, λ Beta protein anneals the oligo to complementary single-strand DNA at the replication fork. Mismatches are created and the methyldirected mismatch repair (MMR) system acts to eliminate the mismatches inhibiting recombination. Three ways to evade MMR through oligo design include, in addition to the desired change 1) a C~C mismatch six bp from that change, 2) four or more adjacent mismatches, or 3) mismatches at four or more consecutive wobble positions. The latter proves useful for making high frequency changes that alter only the target amino-acid sequence and even allows modification of essential genes. Efficient uptake of DNA is important for oligo-mediated recombination. Uptake of oligos or plasmids is growth media-dependent and is 10,000-fold reduced for cells grown in minimal vs rich medium. Genome-wide engineering technologies utilizing recombineering will benefit from both optimized recombination frequencies and a greater understanding of how biological processes such as DNA replication and cell division impact recombinants formed at multiple chromosomal loci. Recombination events at multiple loci in individual cells are described here.

Background:The domains in RNA polymerase involved in elongation slippage are unknown. Results: We isolated E. coli RNA polymerase rpoB mutants with altered transcriptional slippage. Conclusion:The fork domain of RNA polymerase controls slippage. Biochemical analysis of the mutants validates the genetic schemes. Significance: Our work sheds light on the mechanism for maintenance of RNA-DNA register during transcription.