The Mus81-Eme1 complex is a structure-selective endonuclease with a critical role in the resolution of recombination intermediates during DNA repair after interstrand cross-links, replication fork collapse, or double-strand breaks. To explain the molecular basis of 3′ flap substrate recognition and cleavage mechanism by Mus81-Eme1, we determined crystal structures of human Mus81-Eme1 bound to various flap DNA substrates. Mus81-Eme1 undergoes gross substrate-induced conformational changes that reveal two key features: (i) a hydrophobic wedge of Mus81 that separates pre- and post-nick duplex DNA and (ii) a “5′ end binding pocket” that hosts the 5′ nicked end of post-nick DNA. These features are crucial for comprehensive protein-DNA interaction, sharp bending of the 3′ flap DNA substrate, and incision strand placement at the active site. While Mus81-Eme1 unexpectedly shares several common features with members of the 5′ flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81-Eme1 preferentially cleaves 3′ flap DNA substrates with 5′ nicked ends.
The structure-selective nuclease Mus81-Eme1 plays key roles in resolving recombination intermediates such as in the repair of DNA interstrand cross-links. Co-crystallization of the human enzyme bound to flap DNA substrates reveals key structural features essential for substrate selection and active site positioning of the incision strand.
- Mus81-Eme1 undergoes a structural transition from a closed state to an open state in response to a DNA binding.
- A specific “5′ end binding pocket” hosts the nicked junction 5′ end of post-nick duplex DNA.
- A hydrophobic wedge separates pre- and post-nick duplex and facilitates bending of a nicked substrate.
- Mus81-Eme1 shares key structural features with 5′ flap nucleases, but with opposite substrate recognition chirality.
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