Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast
AUTOR(ES)
Storici, Francesca
FONTE
National Academy of Sciences
RESUMO
The repair of chromosomal double-strand breaks (DSBs) can be accomplished through homologous recombination in most organisms. We report here that exogenous oligonucleotides can efficiently target for repair a single DSB induced in a chromosome of yeast. The efficiency of recombinational targeting leading to a desired DNA change can be as high as 20% of cells. The DSB was generated either by a regulatable I-SceI endonuclease just before transformation or appeared spontaneously at the site of a long inverted repeat composed of human Alu sequences. The approach used features of our previously described delitto perfetto system for selecting transformants with integrative recombinant oligonucleotides. The DSB repair mediated by pairs of complementary integrative recombinant oligonucleotides was efficient for targeting to homologous sequences that were close to or distant from the DSB and in the presence of a competing homologous chromosome in diploid cells. We also demonstrate that a DSB can strongly stimulate recombination with single-stranded DNA, without strand bias. These findings expand current models of DSB repair. In addition, we establish a high-throughput system for rapid genome-wide modification with oligonucleotides.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=299876Documentos Relacionados
- Repair of site-specific double-strand breaks in a mammalian chromosome by homologous and illegitimate recombination.
- Lethality induced by a single site-specific double-strand break in a dispensable yeast plasmid.
- Pathway utilization in response to a site-specific DNA double-strand break in fission yeast
- Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli: a method for site-specific mutagenesis.
- DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair.