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

Abstract

MUS81 shares a high-degree homology with the catalytic XPF subunit of the XPF–ERCC1 endonuclease complex. It is catalytically active only when complexed with the regulatory subunits Mms4 or Eme1 in budding and fission yeasts, respectively, and EME1 or EME2 in humans. Although Mus81 complexes are implicated in the resolution of recombination intermediates in vivo, recombinant yeast Mus81-Mms4 and human MUS81-EME1 isolated from Escherichia coli fail to cleave intact Holliday junctions (HJs) in vitro. In this study, we show that human recombinant MUS81-EME2 isolated from E. coli cleaves HJs relatively efficiently, compared to MUS81-EME1. Furthermore, MUS81-EME2 catalyzed cleavage of nicked and gapped duplex deoxyribonucleic acids (DNAs), generating double-strand breaks. The presence of a 5′ phosphate terminus at nicks and gaps rendered DNA significantly less susceptible to the cleavage by MUS81-EME2 than its absence, raising the possibility that this activity could play a role in channeling damaged DNA duplexes that are not readily repaired into the recombinational repair pathways. Significant differences in substrate specificity observed with unmodified forms of MUS81-EME1 and MUS81-EME2 suggest that they play related but non-overlapping roles in DNA transactions.

Control of Mus81 nuclease during the cell cycle

“…This second result is also different from what has been described for budding yeast Mus81‐Mms4, which is maximally active towards model RFs when immunoprecipitated from cells synchronized in mitosis . While more work will be required to clarify this apparent discrepancy, it is important to consider that human MUS81 binds not only EME1 but also a second orthologs of Mms4, EME2, which is known to enhance the activity and decrease the specificity of MUS81 . Hence, high activity of MUS81 immunoprecipitates towards model RFs during S‐phase may simply reflect the constitutive association with EME2, which could conceal the fluctuating activity of MUS81‐EME1‐SLX4.…”

“…One surprising finding arising from the analysis of chromosome breakage upon inhibition of WEE1 was its exquisite dependency on EME2. As discussed above, a key difference between MUS81‐EME2 and MUS81‐EME1 is that MUS81‐EME2 has weaker substrate specificity in vitro and overall higher nuclease activity . A second difference is cellular abundance in cells: EME2 is expressed at lower levels and only ~ 20% of all MUS81 associated with EME2, while the remaining 80% bind EME1 .…”

SLX4-SLX1 Protein-independent Down-regulation of MUS81-EME1 Protein by HIV-1 Viral Protein R (Vpr)

“…The C-terminal regions of EME1 (total of 570 residues) and EME2 (total of 379 residues) are highly homologous with more than 60% sequence similarity in human (43,44). Multiple studies have suggested that these complexes are involved in maintenance of genomic stability and damaged DNA processing (43)(44)(45)(46)(47). Purified recombinant MUS81-EME1 recognizes and preferentially cleaves several DNA structures, including 3Ј-flap, aberrant replication forks, and nicked Holliday junctions (45,48).…”

Holliday junction processing enzymes as guardians of genome stability

“…EME1 is nonetheless required for the stability and activity of the MUS81-EME1 complex, and acts as a regulatory subunit. MUS81 also interacts with EME2, to form the MUS81-EME2 nuclease [27][28][29], but this appears to be dispensable for the resolution of recombination intermediates [30]. Ti BS Figure 1.…”