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Bacterial DNA polymerases participate in oligonucleotide recombination
Author(s) -
Li Xintian,
Thomason Lynn C.,
Sawitzke James A.,
Costantino Nina,
Court Donald L.
Publication year - 2013
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.12231
Subject(s) - biology , recombineering , homologous recombination , genetics , flp frt recombination , oligonucleotide , recombination , dna polymerase , dna , in vitro recombination , genetic recombination , microbiology and biotechnology , complementary dna , gene , molecular cloning
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 E scherichia coli functions that process the recombining oligo and affect bacteriophage λ R ed‐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 DN A 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 ( P ol I and P ol III ) and DNA ligase are directly involved with oligo recombination.