Site-specific DNA double-strand break generated by I-SceI endonuclease enhances ectopic homologous recombination in Pyricularia oryzae

Takayuki Arazoe, Tetsuya Younomaru, Shuichi Ohsato, Makoto Kimura, Tsutomu Arie, Shigeru Kuwata

Research output: Contribution to journalLetter

9 Citations (Scopus)

Abstract

To evaluate the contribution of DNA double-strand breaks (DSBs) to somatic homologous recombination (HR) in Pyricularia oryzae, we established a novel detection/selection system of DSBs-mediated ectopic HR. This system consists of donor and recipient nonfunctional yellow fluorescent protein (YFP)/blasticidin S deaminase (BSD) fusion genes and the yeast endonuclease I-SceI gene as a recipient-specific DSB inducer. The system enables to detect and select ectopic HR events by the restoration of YFP fluorescence and blasticidin S resistance. The transformed lines with donor and recipient showed low frequencies of endogenous ectopic HR (> 2.1%). Compared with spontaneous HR, c. 20-fold increases in HR and absolute frequency of HR as high as 40% were obtained by integration of I-SceI gene, indicating that I-SceI-mediated DSB was efficiently repaired via ectopic HR. Furthermore, to validate the impact of DSB on targeted gene replacement (TGR), the transformed lines with a recipient gene were transfected with an exogenous donor plasmid in combination with the DSB inducer. TGR events were not observed without the DSB inducer, whereas hundreds of colonies resulting from TGR events were obtained with the DSB inducer. These results clearly demonstrated that the introduction of site-specific DSB promotes ectopic HR repair in P. oryzae.

Original languageEnglish
Pages (from-to)221-229
Number of pages9
JournalFEMS Microbiology Letters
Volume352
Issue number2
DOIs
Publication statusPublished - Mar 2014

Fingerprint

Double-Stranded DNA Breaks
Deoxyribonuclease I
Homologous Recombination
Genes
blasticidin S deaminase
Protein S
Recombinational DNA Repair
Oryza
Gene Fusion
Plasmids
Yeasts
Fluorescence

Keywords

  • DNA double-strand breaks
  • DNA repair
  • Homologous recombination

Cite this

Arazoe, Takayuki ; Younomaru, Tetsuya ; Ohsato, Shuichi ; Kimura, Makoto ; Arie, Tsutomu ; Kuwata, Shigeru. / Site-specific DNA double-strand break generated by I-SceI endonuclease enhances ectopic homologous recombination in Pyricularia oryzae. In: FEMS Microbiology Letters. 2014 ; Vol. 352, No. 2. pp. 221-229.
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abstract = "To evaluate the contribution of DNA double-strand breaks (DSBs) to somatic homologous recombination (HR) in Pyricularia oryzae, we established a novel detection/selection system of DSBs-mediated ectopic HR. This system consists of donor and recipient nonfunctional yellow fluorescent protein (YFP)/blasticidin S deaminase (BSD) fusion genes and the yeast endonuclease I-SceI gene as a recipient-specific DSB inducer. The system enables to detect and select ectopic HR events by the restoration of YFP fluorescence and blasticidin S resistance. The transformed lines with donor and recipient showed low frequencies of endogenous ectopic HR (> 2.1{\%}). Compared with spontaneous HR, c. 20-fold increases in HR and absolute frequency of HR as high as 40{\%} were obtained by integration of I-SceI gene, indicating that I-SceI-mediated DSB was efficiently repaired via ectopic HR. Furthermore, to validate the impact of DSB on targeted gene replacement (TGR), the transformed lines with a recipient gene were transfected with an exogenous donor plasmid in combination with the DSB inducer. TGR events were not observed without the DSB inducer, whereas hundreds of colonies resulting from TGR events were obtained with the DSB inducer. These results clearly demonstrated that the introduction of site-specific DSB promotes ectopic HR repair in P. oryzae.",
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Site-specific DNA double-strand break generated by I-SceI endonuclease enhances ectopic homologous recombination in Pyricularia oryzae. / Arazoe, Takayuki; Younomaru, Tetsuya; Ohsato, Shuichi; Kimura, Makoto; Arie, Tsutomu; Kuwata, Shigeru.

In: FEMS Microbiology Letters, Vol. 352, No. 2, 03.2014, p. 221-229.

Research output: Contribution to journalLetter

TY - JOUR

T1 - Site-specific DNA double-strand break generated by I-SceI endonuclease enhances ectopic homologous recombination in Pyricularia oryzae

AU - Arazoe, Takayuki

AU - Younomaru, Tetsuya

AU - Ohsato, Shuichi

AU - Kimura, Makoto

AU - Arie, Tsutomu

AU - Kuwata, Shigeru

PY - 2014/3

Y1 - 2014/3

N2 - To evaluate the contribution of DNA double-strand breaks (DSBs) to somatic homologous recombination (HR) in Pyricularia oryzae, we established a novel detection/selection system of DSBs-mediated ectopic HR. This system consists of donor and recipient nonfunctional yellow fluorescent protein (YFP)/blasticidin S deaminase (BSD) fusion genes and the yeast endonuclease I-SceI gene as a recipient-specific DSB inducer. The system enables to detect and select ectopic HR events by the restoration of YFP fluorescence and blasticidin S resistance. The transformed lines with donor and recipient showed low frequencies of endogenous ectopic HR (> 2.1%). Compared with spontaneous HR, c. 20-fold increases in HR and absolute frequency of HR as high as 40% were obtained by integration of I-SceI gene, indicating that I-SceI-mediated DSB was efficiently repaired via ectopic HR. Furthermore, to validate the impact of DSB on targeted gene replacement (TGR), the transformed lines with a recipient gene were transfected with an exogenous donor plasmid in combination with the DSB inducer. TGR events were not observed without the DSB inducer, whereas hundreds of colonies resulting from TGR events were obtained with the DSB inducer. These results clearly demonstrated that the introduction of site-specific DSB promotes ectopic HR repair in P. oryzae.

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KW - DNA double-strand breaks

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