Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair  

Abstract

DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.

A family portrait: structural comparison of the Whirly proteins fromArabidopsis thalianaandSolanum tuberosum

“…The crystal structures of the Solanum tuberosum (potato) WHY1 and WHY2 proteins were solved in the free form (Desveaux et al, 2002;Cappadocia et al, 2008Cappadocia et al, , 2010. These structures revealed that both chloroplast-directed and mitochondria-directed Whirly proteins assemble into tetramers with C4 symmetry.…”

“…Also, the treatment of Arabidopsis plants with ciprofloxacin, an inhibitor of DNA gyrases that induces DSBs in both plastids and mitochondria (Rowan et al, 2010;Parent et al, 2011), results in an increase in microhomologymediated DNA rearrangements in the plastids of plants lacking WHY1 and WHY3 (Cappadocia et al, 2010). WHY2, the Whirly protein directed to mitochondria in Arabidopsis, also appears to be involved in repressing the MMBIR pathway in the mitochondria, although to a lesser extent (Cappadocia et al, 2010).…”

“…Whirly proteins are mainly found in the plant kingdom, where they localize to either chloroplasts (specialized mature plastids responsible for photosynthesis) or mitochondria (reviewed in Desveaux et al, 2005). In solution, the recombinant proteins form tetramers (Desveaux et al, 2002;Cappadocia et al, 2010) that bind single-stranded DNA with low sequence specificity (Cappadocia et al, 2010). In the absence of the chloroplast-directed Whirly proteins, the plastid genome of both Arabidopsis thaliana and Zea mays (maize) becomes unstable and accumulates DNA rearrangements that contain microhomologies at their endpoint junction (Maré chal et al, 2009).…”

“…Under some conditions, however, the DNA-repair machinery is unable to cope with all DNA damage and non-conservative repairs occur (Abdelnoor et al, 2003;Cappadocia et al, 2010;Kwon et al, 2010;Maré chal et al, 2009;Shedge et al, 2007). Whirly proteins are negative regulators of a non-conservative repair pathway named microhomology-mediated break-induced replication (MMBIR; Maré chal et al, 2009;Cappadocia et al, 2010).…”

The functions of WHIRLY1 and REDOX-RESPONSIVE TRANSCRIPTION FACTOR 1 in cross tolerance responses in plants: a hypothesis

“…Analyses of the crystal structure of WHIRLY proteins revealed that they form tetramers [68] and that these tetramers can also assemble into 24-mers [69,70]. The 24-oligomers of WHIRLY1 have been isolated from chloroplasts [70] and they are likely to be the structures that bind to the thylakoids.…”