Insertional mutagenesis in Chlamydomonas reinhardtii: An effective strategy for the identification of new genes involved in the DNA damage response.

The formation of double-strand breaks in DNA represents a serious stress for all types of organisms and requires a precisely regulated and organized DNA damage response (DDR) to maintain genetic information and genome integrity. Chlamydomonas reinhardtii possesses the characteristics of both plants and animals and is therefore suitable for the identification of novel genes connected to a wide spectrum of metabolic pathways, including DDR. One very effective tool for the detection and subsequent characterization of new mutants in C. reinhardtii is insertional mutagenesis. We isolated several insertion mutants sensitive to DNA-damaging agents that had disrupted or completely deleted genes with putative functions in the DDR. In most of the analysed mutants, we identified various changes at both ends and even inside the inserted cassette. Using recent information from databases, we were also able to supplement the characteristics of the previously described mutant with a pleiotropic phenotype. In addition, we confirmed the effectiveness of hairpin-PCR as a strategy for the identification of insertion flanking sites and as a tool for the detection of changes at the site of insertion, thus enabling a better understanding of insertion events.