Studies of an Mre11 separation of function mutation give insight into the mechanism of double stranded DNA damage repair

Abstract

The MRE11, RAD50, and NBS1 genes encode the proteins of the Mre11–Rad50–Nbs1 (MRN) complex, which is critical for proper maintenance of genomic integrity. The MRN complex acts in DNA double‐strand break repair (DSBR) detection, repair, and signaling. Exo‐ and endonuclease activities of Mre11 may regulate the choice between non‐homologous end‐joining (NHEJ) versus homologous recombination (HR) repair pathways but the exact mechanism is still nebulous. Two Mre11 mutations have been previously described as separation of function mutants: P. furiosus (Pf) H85S is both exo‐ and endonuclease inactive while Pf Mre11 H52S is exonuclease inactive but endonuclease active. Here we describe a new separation of function mutant: Pf Mre11 Y187C, which is also exonuclease inactive and endonuclease active. We have examined the possible causes for the difference in activities in Pf Mre11 Y187C and for the separation of function phenotype in general. Nuclear Magnetic Resonance (NMR) spectroscopy shows that both Y187C and H52S mutations alter the dynamic landscape of Mre11 affecting the DNA binding properties of the protein. We found that there is no change in binding of AMP (a nucleolytic product) by Y187C compared to WT. For DNA binding, Mre11ND constructs showed that Y187C binds both single and double stranded DNA with lower affinity than WT, but once Rad50 is added to the complex, there was no significant difference in affinity. Single site mutations in S. cerevisiae (yeast) established that the analogous Mre11 mutations are capable of DNA damage repair (i.e., survivability on plates with DNA‐damaging drugs), NHEJ repair, and DNA end resection, which is expected based on the previously described in vivo activity studies of MRN/X. Together, our data reveal that an aromatic group at amino acid position 187 in Pf Mre11 is important for nuclease activity, and our yeast data suggest that the presence of Mre11, but not it’s nuclease activity, is required for DSB repair in both repair pathways.Support or Funding InformationCancer Prevention and Research Institute of Texas (CPRIT)Y187C mutant is exonuclease inactiveFigure 1Affinity for dsDNA binding is lower with Y187C mutationFigure 2