The MSH1 gene in Arabidopsis thaliana (AtMSH1) encodes a modular enzyme that consists of an N-terminal MutS DNA mismatch repair module fused to a C-terminal GIYYIG nuclease. Disruption of MSH1 reverses the low organellar mutation rates that distinguish plants from other eukaryotes. However, the precise mechanism by which MSH1 prevents the accumulation of mutations remains unclear. Here, we show that AtMsh1 accurately recognizes and cleaves dsDNA containing mismatches and short indels. AtMsh1 efficiently cleaves dsDNA containing lesions generated by oxidative damage or deamination, with a strong preference for U:G mismatches. AtMsh1 cleaves DNA through an ATP-dependent enzymatic mechanism that requires divalent metal cofactors such as magnesium. The enzyme introduces incisions at defined positions relative to the lesion or mismatch: approximately nine nucleotides 5 prime of the mismatch on the affected strand and twelve nucleotides 3 prime on the complementary strand. This offset cleavage generates staggered DNA ends with three-nucleotide overhangs. Although AtMsh1 displays positional specificity in its cleavage activity on substrates containing lesions and mismatches, it exhibits nonspecific double-stranded DNA cleavage in the presence of manganese;. These findings establish AtMsh1 as a minimal mismatch repair (MMR) system in which mismatch/lesion recognition and DNA cleavage are functionally coupled. We propose that the resulting dsDNA breaks are processed by exonucleases that mediate single-stranded DNA resection, thereby removing the mismatch or lesion while generating a 3' single-stranded DNA overhang suitable for homologous recombination (HR) repair and gene conversion.
Penafiel-Ayala, A., Zhou, C., Baruch-Torres, N., Sloan, D. B., Arimura, S.-i., Brieba, L. G.
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