Plant MutS Homolog 1 is a mismatch-directed nuclease required for organelle genome maintenance

ABSTRACT The exceptionally low mutation rates of plant organellar genomes imply the existence of DNA surveillance mechanisms that counteract replication errors and DNA damage. Genetic evidence implicates MutS HOMOLOG 1 (MSH1) as a central component of this pathway, as loss of MSH1 results in the accumulation of point mutations. MSH1 is a unique protein that combines an N-terminal MutS-like mismatch-recognition module with a C-terminal GIY–YIG nuclease domain. Here, we show that Arabidopsis thaliana MSH1 (AtMsh1) recognizes mismatches, insertion/deletion loops, and damaged bases within double-stranded DNA and introduces staggered DNA breaks at positions flanking the mismatch or lesion. Given the presence of an active homologous recombination machinery in plant organelles, we hypothesize that these DNA ends may be processed by exonucleases to remove the mismatched or damaged DNA while generating 3′ single-stranded DNA substrates suitable for homologous recombination-mediated repair and gene conversion. Together, our findings support a model in which AtMsh1 functions as a minimal mismatch repair system that couples mismatch recognition to DNA incision, providing a potential mechanism for suppressing mutation accumulation and maintaining the remarkable stability of plant organellar genomes. Key Points AtMsh1 recognizes and cleaves DNA mismatches, short insertion/deletion loops, and damaged DNA. AtMsh1 functions as a minimal mismatch repair system by coupling mismatch and lesion recognition to double-stranded DNA cleavage. The staggered 3′ single-stranded DNA ends generated by AtMsh1 cleavage are substrates for homologous recombination-mediated repair and gene conversion. Graphical Abstract Mismatch recognition and nuclease activity by plant organellar MutS Homolog 1 drive organellar genome maintenance.