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The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens

The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent path...

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Autores principales: Kamisugi, Yasuko, Whitaker, John W., Cuming, Andrew C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990234/
https://www.ncbi.nlm.nih.gov/pubmed/27537368
http://dx.doi.org/10.1371/journal.pone.0161204
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author Kamisugi, Yasuko
Whitaker, John W.
Cuming, Andrew C.
author_facet Kamisugi, Yasuko
Whitaker, John W.
Cuming, Andrew C.
author_sort Kamisugi, Yasuko
collection PubMed
description The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G(2) of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency.
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spelling pubmed-49902342016-08-29 The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens Kamisugi, Yasuko Whitaker, John W. Cuming, Andrew C. PLoS One Research Article The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G(2) of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency. Public Library of Science 2016-08-18 /pmc/articles/PMC4990234/ /pubmed/27537368 http://dx.doi.org/10.1371/journal.pone.0161204 Text en © 2016 Kamisugi et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Kamisugi, Yasuko
Whitaker, John W.
Cuming, Andrew C.
The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens
title The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens
title_full The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens
title_fullStr The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens
title_full_unstemmed The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens
title_short The Transcriptional Response to DNA-Double-Strand Breaks in Physcomitrella patens
title_sort transcriptional response to dna-double-strand breaks in physcomitrella patens
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990234/
https://www.ncbi.nlm.nih.gov/pubmed/27537368
http://dx.doi.org/10.1371/journal.pone.0161204
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