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

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

Molecular characterization of <i>aph7</i>″ insertions.

<p>(A) Southern analysis of <i>aph7</i>″ insertions in individual mutants. The restriction enzymes used for each experiment are indicated; positions of the restriction sites within the <i>aph7</i>″ construct probe used for hybridization are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105482#pone-0105482-g001" target="_blank">Figure 1A</a>. (B) Structure of the hairpin adaptor with a blunt end; examples of hairpins with ends compatible with selected restriction enzymes are indicated. (C) PCR products, separated in agarose gels and stained with ethidium bromide, that were generated after two rounds of nested PCR from a genomic library produced by <i>Pst</i>I digest.</p

Transformation efficiency of <i>C. reinhardtii</i> mutants.

<p>(A) Transformation efficiency calculated as the frequency of Arg-prototrophic transformants per a total number of transformed cells normalized to 1 pmol of pUCARG7.8 construct used for transformation. (B) Efficiency of homology-driven integration estimated as a ratio between the transformation efficiencies obtained with the pUCBM20ΔARG and pUCARG7.8 constructs. (A, B) C = <i>cw15-302 arg2</i>; standard errors of three independent experiments are indicated (N = 3). (C) Growth curves of the Z12 strain and selected Z12 Arg-prototrophic revertants in TAP liquid media with or without Arg. <i>cw15-302 arg2</i> cells complemented with the <i>ARG7</i> construct were used as an Arg-prototrophic control (C-ARG7).</p

Structure of insertion sites.

<p>Black arrows represent the aph7″ insert, red arrows indicate homologues to known DNA repair genes, blue arrows represent genes unrelated to DNA repair, and green boxes depict DNA fragments that were co-transformed to the insertion sites from ectopic locations. Sequences at the insert borders are indicated.</p

Sensitivity of <i>C. reinhardtii</i> mutant strains to genotoxic treatments.

<p>(A) Structure of the <i>aph7"</i> construct used for the insertional mutagenesis. Primers (arrows), restriction sites and the region used as a probe for Southern hybridization are indicated. (B) Growth curves of mutant and the parental <i>cw15-302 arg2</i> strains (denoted as C) in TAP liquid media supplemented with zeocin, MMC or MMS. (C) Growth of mutant strains on TAP plates exposed to increasing dose of UV-C or HU. Pictures were taken five days after inoculation.</p

Relative sensitivity to genotoxic treatments in respect to other mutants and the control strain <i>cw15-302 arg2</i> (+++ high sensitivity, ++ intermediate sensitivity, + mild sensitivity, −no sensitivity).

<p>Relative sensitivity to genotoxic treatments in respect to other mutants and the control strain <i>cw15-302 arg2</i> (+++ high sensitivity, ++ intermediate sensitivity, + mild sensitivity, −no sensitivity).</p