Biochemical and structural characterization of analogs of MRE11 breast cancer-associated mutant F237C

Abstract

The MRE11–RAD50–NBS1 (MRN) protein complex plays a vital role in DNA double strand break sensing, signaling, and repair. Mutation in any component of this complex may lead to disease as disrupting DNA double strand break repair has the potential to cause translocations and loss of genomic information. Here, we have investigated an MRE11 mutation, F237C, identified in a breast cancer tumor. We found that the analogous mutant of Pyrococcus furiosus Mre11 diminishes both the exonuclease and endonuclease activities of Mre11 in vitro. Solution state NMR experiments show that this mutant causes structural changes in the DNA-bound Mre11 for both exo- and endonuclease substrates and causes the protein to become generally more rigid. Moreover, by comparing the NMR data for this cancer-associated mutant with two previously described Mre11 separation-of-nuclease function mutants, a potential allosteric network was detected within Mre11 that connects the active site to regions responsible for recognizing the DNA ends and for dimerization. Together, our data further highlight the dynamics required for Mre11 nuclease function and illuminate the presence of allostery within the enzyme.

LRET-derived HADDOCK structural models describe the conformational heterogeneity required for DNA cleavage by the Mre11-Rad50 DNA damage repair complex

“…H17E is, however, a competent ssDNA endonuclease, showing nearly twice the activity of WT MR on the Cy3/BHQ2 ssDNA substrate in the absence of ATP (Figure 3D). This result is consistent with previous reports that dsDNA and ssDNA substrates use different binding sites on Mre11 (Rahman et al, 2021(Rahman et al, , 2020. When ATP is added, the ssDNA endonuclease activity of MR H17E decreases to WT levels.…”

Dynamic Properties of the DNA Damage Response Mre11/Rad50 Complex

“…The S. cerevisiae Mre11 R10T mutant, affecting the nuclease domain, showed altered dynamics in the capping domain in the MD simulations [40], revealing a complex dynamic relationship between the nuclease domain and the capping domain. A similar relationship was characterized by the NMR chemical shift analyses in mutants affected by a single amino acid substitution corresponding to a missense mutation found in cancer in human Mre11 [41,42]. The movement of the capping domain can be connected to the ability of Mre11/Rad50 complexes to facilitate the unwinding of the DNA end [43].…”

“…shift analyses in mutants affected by a single amino acid substitution corresponding to a missense mutation found in cancer in human Mre11 [41,42]. The movement of the capping domain can be connected to the ability of Mre11/Rad50 complexes to facilitate the unwinding of the DNA end [43].…”

Exonucleases: Degrading DNA to Deal with Genome Damage, Cell Death, Inflammation and Cancer

“…Its exonuclease activity plays an essential role in DDR, degrading DNA between the endonucleolytic incision sites, which creates an entry site for the long-range resection nucleases [ 153 ]. Mutations in MRE11A have been found in some types of cancer characterized by chromosomal instabilities such as breast, endometrium, and colon [ 154 , 155 , 156 , 157 ]. Mutations Y187C and H52S inactivate MRE11A exonuclease but not endonuclease activity [ 158 ].…”