Crystal structures of the structure-selective nuclease Mus81-Eme1 bound to flap DNA substrates

Abstract

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 0 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 0 end binding pocket that hosts the 5 0 nicked end of post-nick DNA. These features are crucial for comprehensive protein-DNA interaction, sharp bending of the 3 0 flap DNA substrate, and incision strand placement at the active site. While Mus81-Eme1 unexpectedly shares several common features with members of the 5 0 flap nuclease family, the combined structural, biochemical, and biophysical analyses explain why Mus81-Eme1 preferentially cleaves 3 0 flap DNA substrates with 5 0 nicked ends.

Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5′ flap DNA: basis of interstrand cross-link repair by FAN1

“…Structural studies on flap structure-selective nucleases such as FEN1/ExoI and MUS81, which preferably incise 59 and 39 flap DNA substrates, respectively, reveal that these nucleases share several common structural features for substrate recognition and cleavage (Orans et al 2011;Tsutakawa et al 2011Tsutakawa et al , 2014Gwon et al 2014). FAN1 also shares conserved features with these nucleases (Fig.…”

“…The significance of this domain is illustrated by functional experiments in fission yeast, in which expression of a truncated FAN1ΔN193 phenocopied a FAN1-deficient strain with respect to its response to a cross-linking agent (Fontebasso et al 2013). The NTD possesses a fully exposed binding site for the 39 nicked end, which is in contrast to the deep binding pocket for the 39 flap in flap endonuclease 1 (FEN1, also known as 3Q8K) (Tsutakawa et al 2011) or for the 59 nicked end in Mus81-Eme1 (MUS81, also known as 4P0S and 4P0R) (Gwon et al 2014). Therefore, the 39 nicked end can be further extended, which explains why FAN1 incises the 59 nicked strand of the 39 flap DNA (Fig.…”

“…5B). Although not directly analogous, Mus81-Eme1 also recognizes the 39 flap substrate through four domains (nuclease, nuclease-like, and two helix-hairpinhelix [HhH 2 ] domains) and undergoes a gross rigid body movement of the HhH 2 domain to place the scissile bond into the active site (Chang et al 2008;Gwon et al 2014). Such rotation has been proposed to provide Mus81-Eme1 with exonuclease activity.…”

“…The human FAN1 (residues 330-1017) was amplified by PCR, inserted into pET28a, and purified by the same protocol used for the purification of PaFAN1. The fulllength human ID complex and human Mus81 (246-551)-Eme1 (178-570) complex were prepared as previously described (Yuan et al 2009;Gwon et al 2014). …”

Resolution of single and double Holliday junction recombination intermediates by GEN1

“…HJs are a poor substrate for MUS81-EME1 and Mus81-Mms4, because both prefer to cleave nicked HJs (46)(47)(48)(49)(50). Because cleavage requires structural transitions at the junction point, it is possible that the nick relaxes the topological constraints that inhibit efficient cleavage of intact HJs.…”