<i>Chlamydomonas</i> chloroplasts can use short dispersed repeats and multiple pathways to repair a double‐strand break in the genome

Odom, Obed W.; Baek, Kwang Hyun; Dani, Radhika; Herrin, David L.

doi:10.1111/j.1365-313x.2007.03376.x

Cited by 36 publications

(29 citation statements)

References 49 publications

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“…The cpDNA of Chlamydomonas is relatively repeat rich (21), and the I-CreII-induced deletions described previously implicated perfect repeats of 15-62 bp, with a strong bias for repeats >30 bp (20). Despite the paucity of such repeats in Arabidopsis, the I-CreII-induced DSB was repaired efficiently, and the sizes of the accompanying deletions were similar to those obtained in Chlamydomonas.…”

Section: Discussionmentioning

confidence: 70%

“…Arabidopsis should be a promising system in which to identify them, however. Previously, the lack of evidence for NHEJ repair in Chlamydomonas (20), or associated with an insertion element in tobacco (37), prompted the suggestion that the absence of this ability might account for the lack of horizontally acquired DNA in green plant chloroplasts. These results weaken that hypothesis, but stop short of negating it, because the NHEJ-like repair events were only a minor fraction of the total.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, we used a homing intron and its endonuclease (I-CreII) to examine DSB repair in the Chlamydomonas chloroplast-evidence of all three HR pathways was obtained, but not for NHEJ or MMEJ (20). In the absence of the intron, repair of the DSB was mostly by SSA between intergenic repeats of 20-60 bp that are abundant in that cp genome (21).…”

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confidence: 99%

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Microhomology-mediated and nonhomologous repair of a double-strand break in the chloroplast genome of Arabidopsis

Kwon

Huq

Herrin

2010

Proc. Natl. Acad. Sci. U.S.A.

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Chloroplast DNA (cpDNA) is under great photooxidative stress, yet its evolution is very conservative compared with nuclear or mitochondrial genomes. It can be expected that DNA repair mechanisms play important roles in cpDNA survival and evolution, but they are poorly understood. To gain insight into how the most severe form of DNA damage, a double-strand break (DSB), is repaired, we have developed an inducible system in Arabidopsis that employs a psbA intron endonuclease from Chlamydomonas, I-CreII, that is targeted to the chloroplast using the rbcS1 transit peptide. In Chlamydomonas, an I-CreII-induced DSB in psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20-60 bp) abundant in that genome; Arabidopsis cpDNA is very repeat poor, however. Phenotypically strong and weak transgenic lines were examined and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of DNA at the psbA locus, but not cpDNA as a whole, in the strongly expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site indicative of DSB repair using microhomology (6-12 bp perfect repeats, or 10-16 bp with mismatches) and no homology. These results provide evidence of alternative DSB repair pathways in the Arabidopsis chloroplast that resemble the nuclear, microhomologymediated and nonhomologous end joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, the data suggest an evolutionary relationship may exist between the repeat structure of the genome and the organelle's ability to repair broken chromosomes.DNA repair | evolution | homing endonuclease | I-CreII | plastid DNA C hloroplast DNA (cpDNA) is closely associated with the photosynthetic membranes that harvest radiant energy and strip electrons from water, producing molecular oxygen as a byproduct (1, 2). Highly reactive forms of oxygen are also generated, and despite the presence of detoxifying enzymes, photooxidative damage is a serious problem. Although there has been extensive study of the damage, protection, and repair of photosynthetic membranes, there has been little attention paid to the genetic consequences of photooxidative stress (3). This can be attributed, at least in part, to a grossly incomplete knowledge of how cpDNA is replicated and maintained throughout the life of a plant (3, 4).However, despite the high-stress environment, the evolution of cpDNA is more conservative than nuclear or mitochondrial DNA (5). We can thus infer that DNA repair mechanisms play important roles in protecting cpDNA and are intimately involved in its evolution. And although some processes have been identified (6-11), the panoply of organelle repair mechanisms is not known, nor is it clear which are most important, or how they have coevolved with the genome. Although we expect that some cpDNA repair processes will reflect its prokaryotic ancestry, the consequences of eukaryotic cell integration...

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Section: Discussionmentioning

confidence: 70%

Section: Discussionmentioning

confidence: 99%

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Microhomology-mediated and nonhomologous repair of a double-strand break in the chloroplast genome of Arabidopsis

Kwon

Huq

Herrin

2010

Proc. Natl. Acad. Sci. U.S.A.

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“…The DNA-maintenance machinery is also required for normal chloroplast function. For example, chloroplast DNA DSB repair by homologous recombination was demonstrated in Chlamydomonas (Odom et al, 2008); and in A. thaliana, a chloroplast-targeted homolog of RecA encoded by the nuclear genome was shown to be involved in chloroplast DSB repair (Rowan et al, 2010). Plant RAD52 homologs targeted to the chloroplast, such as A. thaliana RAD52-2, could take part in the chloroplast DNA repair pathway.…”

Section: Discussionmentioning

confidence: 99%

Identification of PlantRAD52Homologs and Characterization of theArabidopsis thaliana RAD52-Like Genes

et al. 2011

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RADiation sensitive52 (RAD52) mediates RAD51 loading onto single-stranded DNA ends, thereby initiating homologous recombination and catalyzing DNA annealing. RAD52 is highly conserved among eukaryotes, including animals and fungi. This article reports that RAD52 homologs are present in all plants whose genomes have undergone extensive sequencing. Computational analyses suggest a very early RAD52 gene duplication, followed by later lineage-specific duplications, during the evolution of higher plants. Plant RAD52 proteins have high sequence similarity to the oligomerization and DNA binding N-terminal domain of RAD52 proteins. Remarkably, the two identified Arabidopsis thaliana RAD52 genes encode four open reading frames (ORFs) through differential splicing, each of which specifically localized to the nucleus, mitochondria, or chloroplast. The A. thaliana RAD52-1A ORF provided partial complementation to the yeast rad52 mutant. A. thaliana mutants and RNA interference lines defective in the expression of RAD52-1 or RAD52-2 showed reduced fertility, sensitivity to mitomycin C, and decreased levels of intrachromosomal recombination compared with the wild type. In summary, computational and experimental analyses provide clear evidence for the presence of functional RAD52 DNA-repair homologs in plants.

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“…A NHEJ repair pathway could be involved in the formation of NHEJ-like events. However, there is little support for the existence of such a repair pathway in plant organelles, particularly in plastids (Odom et al, 2008;Kohl and Bock, 2009). Furthermore, whereas MHMRs are detected in the absence or presence of ciprofloxacin, all but one NHEJ-like event are detected in ciprofloxacin-treated plants, thus raising the possibility that these events could be by-products of the ciprofloxacin-mediated DNA gyrase inhibition (Marvo et al, 1983).…”

Section: Dsbs Can Be Repaired Through a Microhomology-mediated Break-mentioning

confidence: 99%

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

et al. 2010

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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.

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Chlamydomonas chloroplasts can use short dispersed repeats and multiple pathways to repair a double‐strand break in the genome

Cited by 36 publications

References 49 publications

Microhomology-mediated and nonhomologous repair of a double-strand break in the chloroplast genome of Arabidopsis

Microhomology-mediated and nonhomologous repair of a double-strand break in the chloroplast genome of Arabidopsis

Identification of PlantRAD52Homologs and Characterization of theArabidopsis thaliana RAD52-Like Genes

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

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