The Mus81‐Mms4 structure‐selective endonuclease undergoes nicked junction‐induced conformational changes to bend its DNA substrates for cleavage (553.1)

Mus81‐Mms4 is a structure‐selective endonuclease that cleaves joint DNA molecules that form during homologous recombination in mitotic and meiotic cells. Here, we demonstrate by kinetic analysis using physically tethered DNA substrates that budding yeast Mus81‐Mms4 requires inherent flexibility in DNA substrates for optimal catalysis. Förster Resonance Energy Transfer (FRET) experiments further reveal that recognition of 3’‐flap and nicked Holliday junction substrates by Mus81‐Mms4 involves induction of a sharp bend with a 100° angle between the two duplex DNA arms. In addition, thiol crosslinking of Mus81‐Mms4 bound to DNA substrates demonstrates that the heterodimer undergoes a conformational change induced by joint DNA molecules with preferred structural properties. The results from all three approaches support a model for catalysis by Mus81‐Mms4 in which initial DNA binding is based on minimal structural requirements and is followed by a rate‐limiting conformational transition of the substrate and protein, which leads to a sharply kinked substrate that may fray the DNA four bp away from the junction point to position the nuclease for cleavage between the fourth and fifth nucleotides. These data suggest that mutually compatible conformational changes of Mus81‐Mms4 and its substrates tailor its incision activity to nicked junction molecules, resulting in products that cannot be directly ligated. Grant Funding Source : Supported by NIH and TRDRP