15 research outputs found
Le maintien de la stabilitĂ© gĂ©nomique du plastide : un petit gĂ©nome dâune grande importance
Chez les plantes, le génome plastidique est continuellement exposé à divers stress mutagÚnes,
tels lâoxydation des bases et le blocage des fourches de rĂ©plication. Ătonnamment, malgrĂ© ces
menaces, le gĂ©nome du plastide est reconnu pour ĂȘtre trĂšs stable, sa stabilitĂ© dĂ©passant mĂȘme celle
du gĂ©nome nuclĂ©aire. NĂ©anmoins, les mĂ©canismes de rĂ©paration de lâADN et du maintien de la
stabilité du génome plastidique sont encore peu connus.
Afin de mieux comprendre ces processus, nous avons développé une approche, basée sur
lâemploi de la ciprofloxacine, qui nous permet dâinduire des bris dâADN double-brins (DSBs)
spĂ©cifiquement dans le gĂ©nome des organelles. En criblant, Ă lâaide de ce composĂ©, une collection de
mutants dâArabidopsis thaliana dĂ©ficients pour des protĂ©ines du nuclĂ©oĂŻde du plastide, nous avons
identifié 16 gÚnes vraisemblablement impliqués dans le maintien de la stabilité génomique de cette
organelle. Parmi ces gĂšnes, ceux de la famille Whirly jouent un rĂŽle primordial dans la protection du
génome plastidique face aux réarrangements dépendants de séquences de microhomologie. Deux
autres familles de gÚnes codant pour des protéines plastidiques, soit celle des polymérases de types-I
et celle des recombinases, semblent davantage impliquées dans les mécanismes conservateurs de
réparation des DSBs. Les relations épistatiques entre ces gÚnes et ceux des Whirly ont permis de
définir les bases moléculaires des mécanismes de la réparation dépendante de microhomologies
(MHMR) dans le plastide. Nous proposons également que ce type de mécanismes servirait en quelque
sorte de roue de secours pour les mécanismes conservateurs de réparation.
Finalement, un criblage non-biaisé, utilisant une collection de plus de 50,000 lignées mutantes
dâArabidopsis, a Ă©tĂ© rĂ©alisĂ©. Ce criblage a permis dâĂ©tablir un lien entre la stabilitĂ© gĂ©nomique et le
métabolisme des espÚces réactives oxygénées (ROS). En effet, la plupart des gÚnes identifiés lors de ce
criblage sont impliqués dans la photosynthÚse et la détoxification des ROS. Globalement, notre étude
a permis dâĂ©largir notre comprĂ©hension des mĂ©canismes du maintien de la stabilitĂ© gĂ©nomique dans
le plastide et de mieux comprendre lâimportance de ces processus.The plant plastidial genome is constantly threatened by many mutagenic stresses, such as
base oxidation and replication fork stalling. Despite these threats, the plastid genome has long been
known to be more stable than the nuclear genome, suggesting that alterations of its structure would
have dramatic consequences on plant fitness. At the moment, little is known about the genes and the
pathways allowing such conservation of the organelle genome sequences.
To gain insight into these mechanisms, we developed an assay which uses ciprofloxacin, a
gyrase inhibitor, to generate DNA double-strand breaks (DSBs) exclusively in plant organelles. By
screening mutants deficient for proteins composing the plastid nucleoid on ciprofloxacin, we were
able to identify 16 candidate genes, most likely involved in the repair of DSBs in plastid. Among these
genes, those of the Whirly family of single-stranded DNA binding proteins are shown to be key factors
in protecting the genome from error-prone microhomology mediated repair (MHMR). Two other
family of proteins, the plastid type-I polymerases and the plastid recombinases, seem to be involved in
the conservative repair pathways. The evaluation of the epistatic relationship between those two
genes and the Whirly genes led us to define the molecular basis of MHMR and to propose that they
might act as a backup system for conservative repair pathways.
Finally, a non-biased screen, using 50,000 different insertion lines, allowed the identification
of numerous genes that were already associated with ROS homeostasis, suggesting a link between
DNA repair and ROS imbalance. Globally, our study shed light on the mechanisms that allow the
maintenance of plastid genome, while explaining the importance of such conservation of the plastid
genome
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 tetramerâtetramer 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
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
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
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
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
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