The plant-specific RNA Polymerase V (Pol V) plays a key role in gene silencing, but its role in repair of double stranded DNA breaks is unclear. Excision of the transposable element mPing creates double stranded breaks that are repaired by NHEJ. We measured mPing excision site repair in multiple DNA methylation mutants including pol V using an mPing : GFP reporter. Two independent mutant alleles of pol V showed less GFP expression, indicating that the Pol V protein plays a role in excision site repair. Sequence analysis of the pol V excision sites indicated an elevated rate of large deletions consistent with less efficient repair. These results clarify the role of Pol V, but not other RNA-directed DNA methylation proteins (Pol IV) or maintenance DNA methylation pathways ( MET1 ), in the repair of double-strand DNA breaks.
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Mitochondrial DNA Repair in an Arabidopsis thaliana Uracil N-Glycosylase Mutant
Substitution rates in plant mitochondrial genes are extremely low, indicating strong selective pressure as well as efficient repair. Plant mitochondria possess base excision repair pathways; however, many repair pathways such as nucleotide excision repair and mismatch repair appear to be absent. In the absence of these pathways, many DNA lesions must be repaired by a different mechanism. To test the hypothesis that double-strand break repair (DSBR) is that mechanism, we maintained independent self-crossing lineages of plants deficient in uracil-N-glycosylase (UNG) for 11 generations to determine the repair outcomes when that pathway is missing. Surprisingly, no single nucleotide polymorphisms (SNPs) were fixed in any line in generation 11. The pattern of heteroplasmic SNPs was also unaltered through 11 generations. When the rate of cytosine deamination was increased by mitochondrial expression of the cytosine deaminase APOBEC3G, there was an increase in heteroplasmic SNPs but only in mature leaves. Clearly, DNA maintenance in reproductive meristem mitochondria is very effective in the absence of UNG while mitochondrial genomes in differentiated tissue are maintained through a different mechanism or not at all. Several genes involved in DSBR are upregulated in the absence of UNG, indicating that double-strand break repair is a general system of repair in plant mitochondria. It is important to note that the developmental stage of tissues is critically important for these types of experiments.
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- Award ID(s):
- 1933590
- PAR ID:
- 10158503
- Date Published:
- Journal Name:
- Plants
- Volume:
- 9
- Issue:
- 2
- ISSN:
- 2223-7747
- Page Range / eLocation ID:
- 261
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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The plant-specific RNA Polymerase V (Pol V) plays a key role in gene silencing, but its role in repair of double stranded DNA breaks is unclear. Excision of the transposable element mPing creates double stranded breaks that are repaired by NHEJ. We measured mPing excision site repair in multiple DNA methylation mutants including pol V using an mPing:GFP reporter. Two independent mutant alleles of pol V showed less GFP expression, indicating that the Pol V protein plays a role in excision site repair. Sequence analysis of the pol V excision sites indicated an elevated rate of large deletions consistent with less efficient repair. These results clarify the role of Pol V, but not other RNA-directed DNA methylation proteins (Pol IV) or maintenance DNA methylation pathways (MET1), in the repair of double-strand DNA breaks.more » « less
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Abstract DNA double-strand breaks require repair or risk corrupting the language of life. To ensure genome integrity and viability, multiple DNA double-strand break repair pathways function in eukaryotes. Two such repair pathways, canonical non-homologous end joining and homologous recombination, have been extensively studied, while other pathways such as microhomology-mediated end joint and single-strand annealing, once thought to serve as back-ups, now appear to play a fundamental role in DNA repair. Here, we review the molecular details and hierarchy of these four DNA repair pathways, and where possible, a comparison for what is known between animal and fungal models. We address the factors contributing to break repair pathway choice, and aim to explore our understanding and knowledge gaps regarding mechanisms and regulation in filamentous pathogens. We additionally discuss how DNA double-strand break repair pathways influence genome engineering results, including unexpected mutation outcomes. Finally, we review the concept of biased genome evolution in filamentous pathogens, and provide a model, termed Biased Variation, that links DNA double-strand break repair pathways with properties of genome evolution. Despite our extensive knowledge for this universal process, there remain many unanswered questions, for which the answers may improve genome engineering and our understanding of genome evolution.
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Mitochondrial and plastid genomes in land plants exhibit some of the slowest rates of sequence evolution observed in any eukaryotic genome, suggesting an exceptional ability to prevent or correct mutations. However, the mechanisms responsible for this extreme fidelity remain unclear. We tested seven candidate genes involved in cytoplasmic DNA replication, recombination, and repair (
POLIA ,POLIB ,MSH1 ,RECA3 ,UNG ,FPG , andOGG1 ) for effects on mutation rates in the model angiospermArabidopsis thaliana by applying a highly accurate DNA sequencing technique (duplex sequencing) that can detect newly arisen mitochondrial and plastid mutations even at low heteroplasmic frequencies. We find that disruptingMSH1 (but not the other candidate genes) leads to massive increases in the frequency of point mutations and small indels and changes to the mutation spectrum in mitochondrial and plastid DNA. We also used droplet digital PCR to show transmission of de novo heteroplasmies across generations inmsh1 mutants, confirming a contribution to heritable mutation rates. This dual-targeted gene is part of an enigmatic lineage within themutS mismatch repair family that we find is also present outside of green plants in multiple eukaryotic groups (stramenopiles, alveolates, haptophytes, and cryptomonads), as well as certain bacteria and viruses.MSH1 has previously been shown to limit ectopic recombination in plant cytoplasmic genomes. Our results point to a broader role in recognition and correction of errors in plant mitochondrial and plastid DNA sequence, leading to greatly suppressed mutation rates perhaps via initiation of double-stranded breaks and repair pathways based on faithful homologous recombination. -
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Abstract Mitochondrial DNA (mtDNA) is indispensable for mitochondrial function and is maintained by DNA repair, turnover, mitochondrial dynamics and mitophagy, along with the inherent redundancy of mtDNA. Base excision repair (BER) is a major DNA repair mechanism in mammalian mitochondria. Mitochondrial BER enzymes are implicated in mtDNA-mediated immune response and inflammation. mtDNA is organized into mitochondrial nucleoids by mitochondrial transcription factor A (TFAM). The regulation of DNA repair activities by TFAM-DNA interactions remains understudied. Here, we demonstrate the modulation of DNA repair enzymes by TFAM concentrations, DNA sequences and DNA modifications. Unlike previously reported inhibitory effects, we observed that human uracil-DNA glycosylase 1 (UNG1) and AP endonuclease I (APE1) have optimal activities at specific TFAM/DNA molar ratios. High TFAM/DNA ratios inhibited other enzymes, OGG1 and AAG. In addition, TFAM reduces the accumulation of certain repair intermediates. Molecular dynamics simulations and DNA-binding experiments demonstrate that the presence of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in certain sequence motifs enhances TFAM-DNA binding, partially explaining the inhibition of OGG1 activity. Bioinformatic analysis of published 8-oxodG, dU, and TFAM-footprint maps reveals a correlation between 8-oxodG and TFAM locations in mtDNA. Collectively, these results highlight the complex regulation of mtDNA repair by DNA sequence, TFAM concentrations, lesions and repair enzymes.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/plants9020261
