High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides
Top Cited Papers
- 29 May 2001
- journal article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 98 (12) , 6742-6746
- https://doi.org/10.1073/pnas.121164898
Abstract
Homologous DNA recombination is a fundamental, regenerative process within living organisms. However, in most organisms, homologous recombination is a rare event, requiring a complex set of reactions and extensive homology. We demonstrate in this paper that Beta protein of phage λ generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs (ssDNA) as short as 30 bases long. This ssDNA recombination can be used to mutagenize or repair the chromosome with efficiencies that generate up to 6% recombinants among treated cells. Mechanistically, it appears that Beta protein, a Rad52-like protein, binds and anneals the ssDNA donor to a complementary single-strand near the DNA replication fork to generate the recombinant. This type of homologous recombination with ssDNA provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes. Beta protein and ssDNA may prove generally applicable for repairing DNA in many organisms.Keywords
This publication has 43 references indexed in Scilit:
- A Highly Efficient Escherichia coli-Based Chromosome Engineering System Adapted for Recombinogenic Targeting and Subcloning of BAC DNAGenomics, 2001
- An efficient recombination system for chromosome engineering in Escherichia coliProceedings of the National Academy of Sciences, 2000
- Rings and filaments of β protein from bacteriophage λ suggest a superfamily of recombination proteinsProceedings of the National Academy of Sciences, 1999
- Rapid modification of bacterial artificial chromosomes by ET- recombinationNucleic Acids Research, 1999
- The beta protein of phage λ binds preferentially to an intermediate in DNA renaturationJournal of Molecular Biology, 1998
- The beta protein of phage λ promotes strand exchangeJournal of Molecular Biology, 1998
- Characterization of the DNA-binding domain of β protein, a component of phage λ Red-pathway, by UV catalyzed cross-linkingGene, 1996
- A simple and efficient method for direct gene deletion inSaccharomyces cerevisiaeNucleic Acids Research, 1993
- Parameters affecting the frequencies of transformation and co‐transfromation with synthetic oligonucleotides in yeastYeast, 1992
- Transformation of yeast with synthetic oligonucleotides.Proceedings of the National Academy of Sciences, 1988