Bacterial DNA polymerases participate in oligonucleotide recombination

Summary Synthetic single‐strand oligonucleotides (oligos) with homology to genomic DNA have proved to be highly effective for constructing designed mutations in targeted genomes, a process referred to as recombineering. The cellular functions important for this type of homologous recombination have...

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Bibliographic Details
Published in:Molecular microbiology 2013-06, Vol.88 (5), p.906-920
Main Authors: Li, Xin‐tian, Thomason, Lynn C., Sawitzke, James A., Costantino, Nina, Court, Donald L.
Format: Article
Language:English
Subjects:
Bacteriophage lambda - genetics
Chromosomes
DNA Ligase ATP
DNA Ligases - metabolism
DNA polymerase
DNA Polymerase I - metabolism
DNA Polymerase III - metabolism
DNA, Bacterial - metabolism
E coli
Escherichia coli
Escherichia coli - enzymology
Escherichia coli - genetics
Genetic markers
Genomics
Oligonucleotides - metabolism
Recombination, Genetic
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Summary:Summary Synthetic single‐strand oligonucleotides (oligos) with homology to genomic DNA have proved to be highly effective for constructing designed mutations in targeted genomes, a process referred to as recombineering. The cellular functions important for this type of homologous recombination have yet to be determined. Towards this end, we have identified Escherichia coli functions that process the recombining oligo and affect bacteriophage λ Red‐mediated oligo recombination. To determine the nature of oligo processing during recombination, each oligo contained multiple nucleotide changes: a single base change allowing recombinant selection, and silent changes serving as genetic markers to determine the extent of oligo processing during the recombination. Such oligos were often not incorporated into the host chromosome intact; many were partially degraded in the process of recombination. The position and number of these silent nucleotide changes within the oligo strongly affect both oligo processing and recombination frequency. Exonucleases, especially those associated with DNA Polymerases I and III, affect inheritance of the silent nucleotide changes in the oligos. We demonstrate for the first time that the major DNA polymerases (Pol I and Pol III) and DNA ligase are directly involved with oligo recombination.
ISSN:0950-382X
1365-2958
1365-2958
DOI:10.1111/mmi.12231