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

Abstract

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.

The Single-Stranded DNA-Binding Protein WHIRLY1 Represses WRKY53 Expression and Delays Leaf Senescence in a Developmental Stage-Dependent Manner in Arabidopsis

“…Experiments with the promoter single-nucleotide mutation further proved that the nucleotides GNNNAAATT of the ERE-like motif and AAAT of the AT-rich region (Yoo et al, 2007) in target genes were crucial for the DNA-binding activity of WHY1, consistent with the analysis of WHY1 protein structure data. In the crystal structure of the WHY1 protein, the DNA ends of the WHY1 downstream promoter sequence are located in close proximity between adjacent 24-mers, thus raising the possibility that two pathways exist for the GNNNAAATT and AAATrich ssDNA in the crystal: one goes to the adjacent promoter of the same tetramer and the other goes to an adjacent 24-mers (Cappadocia et al, 2012). Similarly, the binding affinity of WHY1 is sensitive to changes in the AT-rich sequence downstream of the ERE motif (Fig.…”

“…WHY1 binding to ssDNA in plastids had a function in the DNA repair of detrimental lesions and prevents the spurious annealing of resected DNA molecules with other regions in the plastid genome (Maréchal et al, 2009;Cappadocia et al, 2010Cappadocia et al, , 2012. However, whether nuclear WHY1 bound to the ssDNA of the WRKY53 promoter has the same DSBrelated function is still unclear, since DSB in the nuclear genome were observed so far only during meiosis (Keeney and Neale, 2006) or under UV radiation and oxidation stress.…”

Mechanism of Dual Targeting of the Phytochrome Signaling Component HEMERA/pTAC12 to Plastids and the Nucleus

“…In the nucleus, Why1 binds to single-stranded telomeric DNA to modulate telomere length homeostasis (Yoo et al, 2007) and acts as a transcriptional regulator for pathogen responsive genes (Desveaux et al, 2000;Xiong et al, 2009). Plastidial Why1 is required for chloroplast biogenesis in maize (Prikryl et al, 2008) and plays a possible role in plastidial genome repair (Cappadocia et al, 2010(Cappadocia et al, , 2012. Interestingly, a fraction of Why1 is also associated with the pTAC complex (Pfalz et al, 2006;Melonek et al, 2010).…”

Modified Clp Protease Complex in the ClpP3 Null Mutant and Consequences for Chloroplast Development and Function in Arabidopsis

“…Plastid pTAC11 is a member of the Whirly family of multifunctional RNA-and DNA-binding proteins located in mitochondria and plastids (Krause et al, 2005). Members of the Whirly family are involved in organelle genome stability and quality control and, perhaps, RNA metabolism (Prikryl et al, 2008;Maréchal et al, 2009;Cappadocia et al, 2012). A SET domaincontaining protein (At5g14260) was also significantly overaccumulating in clpp3-1; the SET domain is frequently found in DNA-interacting proteins.…”