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

Andrea Plecenikova,Karel Riha,Miroslava Slaninova

doi:10.1371/journal.pone.0105482

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.

Highlights

DNA double strand breaks (DSBs) pose a serious threat to genome integrity as their erroneous repair may lead to chromosomal rearrangements with potentially lethal consequences for an organism
The use of forward genetic screens for discovery of genes involved DNA repair and DNA damage response has been limited in plants; most plant DDR/DNA repair proteins were identified based on sequence homology with their yeast and mammalian counterparts
SOG1, a transcription factor governing DDR is a plant specific protein that was discovered in a screen to identify suppressors of the radiosensitivity of a nucleotide excision repair mutant [33]

Summary

Introduction

DNA double strand breaks (DSBs) pose a serious threat to genome integrity as their erroneous repair may lead to chromosomal rearrangements with potentially lethal consequences for an organism. The response to DSBs elicits a highly organized and complex cellular program, called the DNA damage response (DDR), which sets in motion processes that mitigate the adverse effects of DNA damage and facilitate DNA repair. Broken DNA is usually repaired by one of two mechanistically distinct pathways: homologous recombination (HR) and non-homologous end joining (NHEJ). The preferred mode of repair and cellular consequences of DDR varies between organisms and is dependant on cell type and cell cycle context [1,2]. While HR is the preferred mode of repair in many unicellular organisms such as budding and fission yeast, NHEJ is the prevalent pathway in plants and animals

Methods

Results

Conclusion