A RAD52‐like single‐stranded DNA binding protein affects mitochondrial DNA repair by recombination

Abstract

SUMMARYThe plant mitochondrial DNA-binding protein ODB1 was identified from a mitochondrial extract after DNAaffinity purification. ODB1 (organellar DNA-binding protein 1) co-purified with WHY2, a mitochondrial member of the WHIRLY family of plant-specific proteins involved in the repair of organellar DNA. The Arabidopsis thaliana ODB1 gene is identical to RAD52-1, which encodes a protein functioning in homologous recombination in the nucleus but additionally localizing to mitochondria. We confirmed the mitochondrial localization of ODB1 by in vitro and in vivo import assays, as well as by immunodetection on Arabidopsis subcellular fractions. In mitochondria, WHY2 and ODB1 were found in large nucleo-protein complexes. Both proteins co-immunoprecipitated in a DNA-dependent manner. In vitro assays confirmed DNA binding by ODB1 and showed that the protein has higher affinity for single-stranded than for double-stranded DNA. ODB1 showed no sequence specificity in vitro. In vivo, DNA co-immunoprecipitation indicated that ODB1 binds sequences throughout the mitochondrial genome. ODB1 promoted annealing of complementary DNA sequences, suggesting a RAD52-like function as a recombination mediator. Arabidopsis odb1 mutants were morphologically indistinguishable from the wild-type, but following DNA damage by genotoxic stress, they showed reduced mitochondrial homologous recombination activity. Under the same conditions, the odb1 mutants showed an increase in illegitimate repair bypasses generated by microhomology-mediated recombination. These observations identify ODB1 as a further component of homologous recombination-dependent DNA repair in plant mitochondria.

Mechanism of Homologous Recombination and Implications for Aging-Related Deletions in Mitochondrial DNA

“…Gualberto and coworkers independently identified Rad52-1B as ODB1 (organellar DNA binding protein 1) based on its DNA binding activity. Rad52-1B/ODB1 shares similar properties with Mgm101, including its preferential binding to ssDNA and the ability to promote single-strand annealing (181). More interestingly, these investigators also found that under genotoxic stress, the recombination in mtDNA repeats is significantly reduced in odb1 mutant plants, reminiscent of the phenotype observed with After the resection of a dsDNA end by a 5=-3= exonuclease, SSB is bound to the exposed ssDNA to prevent the formation of secondary structures.…”

The Mitochondrial DNA (mtDNA)-Associated Protein SWIB5 Influences mtDNA Architecture and Homologous Recombination

“…Treating plants with genotoxic chemicals such as BLM or CIP causes the accumulation of IR products in mitochondrial genomes (Parent et al, 2011;Janicka et al, 2012;Miller-Messmer et al, 2012;Wallet et al, 2015; Figure 1B). Using qPCR, we quantified the differential accumulation of all possible crossover products for 18 repeated regions in 14-d-old seedlings germinated on medium supplemented with 0.75 mM CIP, compared with plants germinated on control medium (Figures 1C to 1E;Supplemental Figure 2; all ANOVA tables are provided in Supplemental Data Set 1).…”

“…Homologous proteins of eubacterial factors involved in HR have been shown to have a role in mtDNA recombination (Miller-Messmer et al, 2012;Wallet et al, 2015). Since plant mitochondrial genomes undergo frequent HR events between repeated regions in contrast to other eukaryotes, plant-specific mediators of organellar homologous recombination such as OSB1, ORGANELLAR DNA BINDING PROTEIN1, and members of the WHIRLY protein family have coevolved in plants (Zaegel et al, 2006;Maréchal et al, 2008;Janicka et al, 2012). However, only a few proteins have been proven to have a role in this process, and more factors remain to be discovered.…”

A Properly Configured Ring Structure Is Critical for the Function of the Mitochondrial DNA Recombination Protein, Mgm101

“…We recently found that the Mgm101 protein in yeast mitochondria share biochemical, structural, and functional similarities with the Rad52-type recombination proteins (10). Similar proteins were also identified in mitochondria from plants and lower animals (10,42,43). Like its bacteriophage counterparts, Mgm101 forms oligomeric rings.…”