Annealing and purification of fluorescently labeled DNA substrates for in vitro assays

Tse, Ying Wah E; Yun, Hwa Young; Wyatt, Haley D.M.

doi:10.1016/j.xpro.2023.102128

STAR Protocols

2023

DOI: 10.1016/j.xpro.2023.102128

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Annealing and purification of fluorescently labeled DNA substrates for in vitro assays

Ying Wah E Tse¹,

Hwa Young Yun²,

Haley D.M. Wyatt³

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2023

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References 33 publications

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Plant Organellar MSH1 Is a Displacement Loop–Specific Endonuclease

Peñafiel-Ayala,

Peralta-Castro,

Mora-Garduño

et al. 2023

Plant and Cell Physiology

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MSH1 is an organellar targeted protein that obstructs ectopic recombination and the accumulation of mutations in plant organellar genomes. MSH1 also modulates the epigenetic status of nuclear DNA, and its absence induces a variety of phenotypic responses. MSH1 is a member of the MutS family of DNA mismatch repair proteins but harbors an additional GIY-YIG nuclease domain that distinguishes it from the rest of this family. How MSH1 hampers recombination and promotes fidelity in organellar DNA inheritance is unknown. Here, we elucidate its enzymatic activities by recombinantly expressing and purifying full-length MSH1 from Arabidopsis thaliana (AtMSH1). AtMSH1 is a metalloenzyme that shows a strong binding affinity for displacement loops (D-loops). The DNA binding abilities of AtMSH1 reside in its MutS domain and not in its GIY-YIG domain, which is the ancillary nickase of AtMSH1. In the presence of divalent metal ions, AtMSH1 selectively executes multiple incisions at D-loops, but not other DNA structures including Holliday junctions or dsDNA, regardless of the presence or absence of mismatches. The selectivity of AtMSH1 to dismantle D-loops supports the role of this enzyme in preventing recombination between short repeats. Our results suggest that plant organelles have evolved novel DNA repair routes centered around the anti-recombinogenic activity of MSH1.

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Plant Organellar MSH1 Is a Displacement Loop–Specific Endonuclease

Peñafiel-Ayala,

Peralta-Castro,

Mora-Garduño

et al. 2023

Plant and Cell Physiology

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show abstract

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Annealing and purification of fluorescently labeled DNA substrates for in vitro assays

Cited by 1 publication

References 33 publications

Plant Organellar MSH1 Is a Displacement Loop–Specific Endonuclease

Plant Organellar MSH1 Is a Displacement Loop–Specific Endonuclease

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