A conserved lysine residue of plant Whirly proteins is necessary for higher order protein assembly and protection against DNA damage

All organisms have evolved specialized DNA repair mechanisms in order to protect their genome against detrimental lesions such as DNA double-strand breaks. In plant organelles, these damages are repaired either through recombination or through a microhomology-mediated break-induced replication pathway. Whirly proteins are modulators of this second pathway in both chloroplasts and mitochondria. In this precise pathway, tetrameric Whirly proteins are believed to bind single-stranded DNA and prevent spurious annealing of resected DNA molecules with other regions in the genome. In this study, we add a new layer of complexity to this model by showing through atomic force microscopy that tetramers of the potato Whirly protein WHY2 further assemble into hexamers of tetramers, or 24-mers, upon binding long DNA molecules. This process depends on tetramer–tetramer interactions mediated by K67, a highly conserved residue among plant Whirly proteins. Mutation of this residue abolishes the formation of 24-mers without affecting the protein structure or the binding to short DNA molecules. Importantly, we show that an Arabidopsis Whirly protein mutated for this lysine is unable to rescue the sensitivity of a Whirly-less mutant plant to a DNA double-strand break inducing agent

Time- and cost-efficient identification of T-DNA insertion sites through targeted genomic sequencing.

Forward genetic screens enable the unbiased identification of genes involved in biological processes. In Arabidopsis, several mutant collections are publicly available, which greatly facilitates such practice. Most of these collections were generated by agrotransformation of a T-DNA at random sites in the plant genome. However, precise mapping of T-DNA insertion sites in mutants isolated from such screens is a laborious and time-consuming task. Here we report a simple, low-cost and time efficient approach to precisely map T-DNA insertions simultaneously in many different mutants. By combining sequence capture, next-generation sequencing and 2D-PCR pooling, we developed a new method that allowed the rapid localization of T-DNA insertion sites in 55 out of 64 mutant plants isolated in a screen for gyrase inhibition hypersensitivity

Overview of Targeted Genomic Sequencing.

<p>Blue rectangles represent genomic DNA, and red rectangles correspond to T-DNA insertions. The grey squares represent the 454 specific primers added in order to bind the sequencing beads (purple circles). The green circles correspond to biotin bound to a red T-DNA specific primer and hybridized to T-DNA. Hybridized sequences are then enriched by capture on streptavidin beads (orange circles).</p

Schematic Illustration of the Insertion Sites in the Three Novobiocin-Sensitive Mutant Lines.

<p>The small black arrows represent the orientation of the CaMV 35S enhancers within the T-DNA (rectangle). For Insertion 3, a different part of the plasmid still containing the enhancer region has been inserted.</p

Coverage of the pSKI015 Vector Obtained by Sequencing.

<p>Features of the pSKI015 are summarized below the coverage graph. The blue rectangles represent the T-DNA cassette with the right (RB) and left (LB) borders in green. The position of the 35 S enhancers are indicated by blue open end arrows. The red lines represent the annealing regions of the three biotinylated primers for each border. The position where the repeated reads align is indicated by the double red arrowhead line on the coverage graph.</p

Forward Genetic Screen to Identify Genes Involved in the Maintenance of Organelle Genome Topology.

<p>Schematic representation of the different steps of the forward genetic screen. Plants with white first true leaves represent the mutants sensitive to ciprofloxacin (CIP) or novobiocin (NOVO).</p

Association of an Insertion Event to a Specific Line by 2D-PCR Pooling.

<p>A. Workflow of the 2D-PCR pooling B. An example of the pooling design for 16 plants. Each plant genomic DNA is pooled in a unique set combination. The plants encompass by the colored rectangle associate to the pool of the same color. C. Data analysis to identify the positive line. All bands on a given gel correspond to the same amplification product in different pools.</p