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Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15748-53. Epub 2003 Dec 12.

Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants.

Author information

  • 1Molecular Control and Genetics Section, Gene Regulation and Chromosome Biology Laboratory, Center for Cancer Research, National Cancer Institute, Building 539, PO Box B, Frederick, MD 21702-1201, USA.

Abstract

Homologous recombination can be used to generate recombinants on episomes or directly on the Escherichia coli chromosome with PCR products or synthetic single-stranded DNA (ssDNA) oligonucleotides (oligos). Such recombination is possible because bacteriophage lambda-encoded functions, called Red, efficiently recombine linear DNA with homologies as short as 20-70 bases. This technology, termed recombineering, provides ways to modify genes and segments of the chromosome as well as to study homologous recombination mechanisms. The Red Beta function, which binds and anneals ssDNA to complementary ssDNA, is able to recombine 70-base oligos with the chromosome. In E. coli, methyl-directed mismatch repair (MMR) can affect these ssDNA recombination events by eliminating the recombinant allele and restoring the original sequence. In so doing, MMR can reduce the apparent recombination frequency by >100-fold. In the absence of MMR, Red-mediated oligo recombination can incorporate a single base change into the chromosome in an unprecedented 25% of cells surviving electroporation. Our results show that Beta is the only bacteriophage function required for this level of recombination and suggest that Beta directs the ssDNA to the replication fork as it passes the target sequence.

PMID:
14673109
[PubMed - indexed for MEDLINE]
PMCID:
PMC307639
Free PMC Article
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