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PLoS One. 2014 Aug 21;9(8):e105482. doi: 10.1371/journal.pone.0105482. eCollection 2014.

Characterization of DNA repair deficient strains of Chlamydomonas reinhardtii generated by insertional mutagenesis.

Author information

1
Gregor Mendel Institute, Austrian Academy of Sciences, Vienna Biocenter (VBC), Vienna, Austria; Department of Genetics, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia.
2
Department of Genetics, Faculty of Natural Sciences, Comenius University in Bratislava, Bratislava, Slovakia.
3
Gregor Mendel Institute, Austrian Academy of Sciences, Vienna Biocenter (VBC), Vienna, Austria; Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic.

Abstract

While the mechanisms governing DNA damage response and repair are fundamentally conserved, cross-kingdom comparisons indicate that they differ in many aspects due to differences in life-styles and developmental strategies. In photosynthetic organisms these differences have not been fully explored because gene-discovery approaches are mainly based on homology searches with known DDR/DNA repair proteins. Here we performed a forward genetic screen in the green algae Chlamydomonas reinhardtii to identify genes deficient in DDR/DNA repair. We isolated five insertional mutants that were sensitive to various genotoxic insults and two of them exhibited altered efficiency of transgene integration. To identify genomic regions disrupted in these mutants, we established a novel adaptor-ligation strategy for the efficient recovery of the insertion flanking sites. Four mutants harbored deletions that involved known DNA repair factors, DNA Pol zeta, DNA Pol theta, SAE2/COM1, and two neighbouring genes encoding ERCC1 and RAD17. Deletion in the last mutant spanned two Chlamydomonas-specific genes with unknown function, demonstrating the utility of this approach for discovering novel factors involved in genome maintenance.

PMID:
25144319
PMCID:
PMC4140758
DOI:
10.1371/journal.pone.0105482
[Indexed for MEDLINE]
Free PMC Article

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