MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes

Abstract

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 thalianaby 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 inmsh1mutants, confirming a contribution to heritable mutation rates. This dual-targeted gene is part of an enigmatic lineage within themutSmismatch 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.MSH1has 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.

Construction of a highly error-prone DNA polymerase for developing organelle mutation systems

“…By comparison, our knowledge of these fundamental processes in the organelles of plants is limited. The evolutionary mutation rates of plant organelle genomes are much lower than those observed in plant nuclear genes ( 2 , 11 , 12 ). To advance our understanding of plant organelle genomes by elevating the mutation rate with mutator DNA polymerases requires the construction and characterisation of error-prone versions of plant organelle DNA polymerases.…”

Mutational Pressure Drives Differential Genome Conservation in Two Bacterial Endosymbionts of Sap-Feeding Insects

“…Separate duplex sequencing libraries were generated for each of the four replicate bacteriome DNA samples (A1, A2, B1, and B2). Duplex library preparation followed protocols described elsewhere ( Wu et al 2020 ). Briefly, samples were fragmented with the Covaris M220 Focused-Ultrasonicator and subsequently end-repaired (NEBNext End Repair Module), A-tailed (Klenow Fragment enzyme, 1 mM dATP), adapter ligated (NEBNext Quick Ligation Module), and treated with a cocktail of three repair enzymes (NEB CutSmart Buffer, Fpg, Uracil-DNA Glycosylase, Endonuclease III).…”

“…Fortunately, the recent advent of several high-fidelity DNA sequencing techniques provides the opportunity to obtain a snapshot of extremely low-frequency SNVs in endosymbiont DNA ( Sloan et al 2018 ). One technique, called duplex sequencing, is particularly suited for such a measurement because it has an exceptionally low error rate of less than 2 × 10 −8 errors per bp ( Kennedy et al 2014 ; Wu et al 2020 ). The high accuracy of this method is achieved by tagging each original DNA fragment with random barcodes and producing multiple sequencing reads from both strands to obtain a consensus sequence.…”

The Dark Side of UV-Induced DNA Lesion Repair

“…Deficiency of AtMSH1 caused incomplete development or premature degeneration of plastids [149]. It has been shown recently that the disruption of the AtMSH1 gene caused a 10-fold and 1000-fold increase in the mutation frequency in mitochondrial and plastid genomes, respectively, indicating an essential role of the encoded protein in controlling the rate of organellar DNA mutagenesis [150]. Finally, mismatched nucleotides in chloroplast can be repaired by gene conversion [151].…”