Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast

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 le...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2003-12, Vol.100 (25), p.14994-14999
Main Authors: Storici, F, Durham, C.L, Gordenin, D.A, Resnick, M.A
Format: Article
Language:English
Subjects:
Binding Sites
Biological Sciences
Chromosomes
Deoxyribonucleases, Type II Site-Specific - chemistry
Deoxyribonucleic acid
Diploidy
DNA
DNA Damage
DNA Mutational Analysis
DNA Repair
DNA, Single-Stranded - genetics
double-strand break repair
Genetic loci
genetic recombination
Genetics
Genome, Fungal
Genomes
integrative recombinant oligonucleotides
Models, Genetic
Nucleotides
Oligonucleotides
Oligonucleotides - chemistry
Plasmids
Plasmids - metabolism
Recombination, Genetic
recombinational repair
Saccharomyces cerevisiae
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae Proteins
Sequence homology
Yeasts
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Summary: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-Scel 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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.2036296100