Human MUS81 complexes stimulate flap endonuclease 1

Abstract

The yeast heterodimeric Mus81-Mms4 complex possesses a structure-specific endonuclease activity that is critical for the restart of stalled replication forks and removal of toxic recombination intermediates. Previously, we reported that Mus81-Mms4 and Rad27 (yeast FEN1, another structure-specific endonuclease) showed mutual stimulation of nuclease activity. In this study, we investigated the interactions between human FEN1 and MUS81-EME1 or MUS81-EME2, the human homologs of the yeast Mus81-Mms4 complex. We found that both MUS81-EME1 and MUS81-EME2 increased the activity of FEN1, but FEN1 did not stimulate the activity of MUS81-EME1 ⁄ EME2. The MUS81 subunit alone and its N-terminal half were able to bind to FEN1 and stimulate its endonuclease activity. A truncated FEN1 fragment lacking the C-terminal region that retained catalytic activity was not stimulated by MUS81. Michaelis-Menten kinetic analysis revealed that MUS81 increased the interaction between FEN1 and its substrates, resulting in increased turnover. We also showed that, after DNA damage in human cells, FEN1 co-localizes with MUS81. These findings indicate that the human proteins and yeast homologs act similarly, except that the human FEN1 does not stimulate the nuclease activities of MUS81-EME1 or MUS81-EME2. Thus, the mammalian MUS81 complexes and FEN1 collaborate to remove the various flap structures that arise during many DNA transactions, including Okazaki fragment processing. Structured digital abstractl FEN1 physically interacts with MUS81 and EME2 by anti tag coimmunoprecipitation (View interaction) l FEN1 physically interacts with MUS81 and EME1 by anti tag coimmunoprecipitation (View interaction) l MUS81 physically interacts with FEN1 by pull down (View interaction) l FEN1 and MUS81 colocalize by fluorescence microscopy (View interaction) l FEN1 physically interacts with MUS81 by anti tag coimmunoprecipitation (View interaction)

Systematic Triple-Mutant Analysis Uncovers Functional Connectivity between Pathways Involved in Chromosome Regulation

“…These data suggest that Mus81 works independently of Mms4 in certain instances. It is interesting to note that in mammals, Mus81 has been shown to pair with two distinct partners, Eme1 and Eme2 (Shin et al, 2012). Although no other partner as been found in budding yeast, our results raise the possibility of a second partner or that in some circumstances, Mus81 can act alone.…”

Human MUS81-EME2 can cleave a variety of DNA structures including intact Holliday junction and nicked duplex

“…The recombinant human MUS81–EME2 complex was purified as previously described (20,32) with the following modifications. The pET21d-MUS81/His-EME2 plasmid was transformed into Escherichia coli BL21-Codon plus (DE3) RIL (Stratagene) and cells (2 l) were grown at 30°C until an A 600 value of 1.0.…”

“…( A ) The purification scheme used to purify the recombinant human MUS81-EME2 complex. The recombinant human MUS81-EME2 was expressed in E. coli and purified using the same procedure as reported (20,32), but modified by the introduction of two additional purification steps (see text for details). P11, Ni 2+ -NTA and heparin indicate phosphocellulose, Ni 2+ -NTA affinity and heparin column chromatography, respectively.…”

Human Fanconi Anemia Complementation Group A Protein Stimulates the 5’ Flap Endonuclease Activity of FEN1

“…RecQ DNA helicases such as Werner syndrome protein WRN and Bloom syndrome protein BLM were shown to stimulate FEN1 through physical interaction [26–28]. It was also reported that MUS81-EME1 and MUS81-EME2, DNA endonucleases involved in interstrand crosslink unhooking and Holliday junction resolution, stimulate FEN1 activity [29]. …”