Repairing DNA Double-Strand Breaks May Improve Genomic Stability by Limiting Organelle-to-Nucleus Gene Transfer

By Pooja Toshniwal PahariaReviewed by Lexie CornerJun 2 2025

A recent study in Nature Plants examined the genetic regulation of endosymbiotic gene transfer (EGT) in plants. Researchers found that deoxyribonucleic acid (DNA) double-strand break repair (DSBR) pathways reduce gene transfer from cell organelles to the nuclear genome.

In contrast, deficiencies in DSBR pathways—specifically in DNA ligase IV (LIG4) and DNA polymerase theta (POLQ) mutants—led to a marked increase in EGT frequency. The study suggests that double-strand breaks (DSBs) in DNA facilitate the integration of organelle DNA (orgDNA) into the nuclear genome.

However, this integration may contribute to transgenerational instability at the neomycin phosphotransferase II (nptII) locus in progeny derived from EGT events. These findings contribute to the understanding of EGT’s implications for plant genetics and biotechnology.

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Understanding Endosymbiotic Gene Transfer

EGT refers to the transfer of genetic material from organelles such as plastids and mitochondria into the nuclear genome. This process is relevant to the evolution of eukaryotic cells from symbiotic relationships and has potential implications for plant biotechnology.

Studying the molecular mechanisms that regulate EGT may improve our understanding of evolutionary biology and inform strategies in genetic engineering.

In eukaryotic cells, nuclear DNA integration involves DSBR pathways. In the model plant Arabidopsis thaliana, non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) pathways repair somatic DSBs and help prevent EGT. However, the extent to which NHEJ and MMEJ influence EGT frequency remains unclear.

About the Study

In this study, researchers investigated the role of DNA double-strand break repair in EGT using Nicotiana tabacum (Petit Havana variety).

The team cultivated tobacco plants under controlled greenhouse conditions, germinating seeds on Murashige and Skoog (MS) medium containing antibiotics such as kanamycin (100–400 mg/l). They conducted regenerative experiments with leaf explants, identifying callus and shoot formation on culture plates. Regenerated shoots were transferred to rooting media for further study.

Guide RNAs (gRNAs) were designed using updated genetic models for targeted genome editing. Genomic DNA from plants was genotyped using polymerase chain reaction (PCR), and transformation vectors were constructed to perform genome editing. CRISPR-associated protein 9 (Cas9) activity was confirmed through Sanger sequencing.

The researchers created loss-of-function mutants of the tobacco homologs of LIG4 and POLQ in mesophyll and egg cells to isolate knockout alleles without visible phenotypes. Mutants with defective DSBR were confirmed through increased sensitivity to bleomycin and assessed for genotoxic stress in seed phenotypes.

Large-scale tissue culture experiments assessed EGT in tobacco explants. Criteria for EGT included shoot or callus regeneration from over 50% of explants within one month and early rooting within three months of primary selection.

"Escapes" referred to plants that failed to regenerate and showed bleaching within one month on kanamycin-containing media. These indicated transient gene transfer events without stable integration. In contrast, stable EGT events involved the nuclear translocation of nptII with orgDNA integration.

Pollen from single-rooted DSBR mutant plants was used to pollinate wild-type flowers to assess EGT in male gametophytes. Kanamycin-resistant seedlings identified after 18 days were considered pollen EGT lines. Genotype effects on EGT frequencies were evaluated using multiple linear regressions.

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Results

The study found that DSBR pathways in eukaryotes limit orgDNA integration into the nuclear genome by reducing DSB availability in both somatic and male germline cells. Pollen-derived EGT events were more frequent in DSBR mutant plants, suggesting that impaired DSBR increases orgDNA integration in the male gametophyte. Similarly, DSBR deficiencies led to higher somatic EGT frequencies.

FGT levels were higher in the male germline compared to somatic tissues or female gametophytes. DSBR-deficient plants exhibited significantly more EGT events than control lines, likely due to the prolonged presence of unrepaired DSBs, which facilitate orgDNA integration. In the male gametophyte, DSBR deficiency increased EGT frequency by 5- to 20-fold.

POLQ mutants showed a 20-fold increase in EGT frequency, highlighting the helicase domain's role in preventing replication-associated DSBs. LIG4 mutants exhibited stronger effects than POLQ mutants, suggesting the NHEJ pathway plays a more prominent role than the TMEJ pathway in preventing EGT.

The findings suggest that DSBR pathways reduce EGT by limiting opportunities for orgDNA to integrate into the nuclear genome. In DSBR-deficient conditions, elevated EGT frequencies could lead to organellar DNA insertions and potential genomic rearrangements, underscoring the importance of maintaining DSBR activity for genome stability.

Journal Reference

Gonzalez-Duran, E., et al. (2025). Suppression of plastid-to-nucleus gene transfer by DNA double-strand break repair. Nat. Plants. DOI: 10.1038/s41477-025-02005-w, https://www.nature.com/articles/s41477-025-02005-w