TY - JOUR
T1 - Mutation of Conserved Mre11 Residues Alter Protein Dynamics to Separate Nuclease Functions
AU - Rahman, Samiur
AU - Beikzadeh, Mahtab
AU - Canny, Marella D.
AU - Kaur, Navneet
AU - Latham, Michael P.
N1 - Publisher Copyright:
© 2020 The Author(s)
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2020/5/1
Y1 - 2020/5/1
N2 - Naked and protein-blocked DNA ends occur naturally during immune cell development, meiosis, and at telomeres as well as from aborted topoisomerase reactions, collapsed replication forks, and other stressors. Damaged DNA ends are dangerous in cells and if left unrepaired can lead to genomic rearrangement, loss of genetic information, and eventually cancer. Mre11 is part of the Mre11–Rad50–Nbs1 complex that recognizes DNA double-strand breaks and has exonuclease and endonuclease activities that help to initiate the repair processes to resolve these broken DNA ends. In fact, these activities are crucial for proper DNA damage repair pathway choice. Here, using Pyrococcus furiosus Mre11, we question how two Mre11 separation-of-function mutants, one previously described but the second first described here, maintain endonuclease activity in the absence of exonuclease activity. To start, we performed solution-state NMR experiments to assign the side-chain methyl groups of the 64-kDa Mre11 nuclease and capping domains, which allowed us to describe the structural differences between Mre11 bound to exo- and endonuclease substrates. Then, through biochemical and biophysical characterization, including NMR structural and dynamics studies, we compared the two mutants and determined that both affect the dynamic features and double-stranded DNA binding properties of Mre11, but in different ways. In total, our results illuminate the structural and dynamic landscape of Mre11 nuclease function.
AB - Naked and protein-blocked DNA ends occur naturally during immune cell development, meiosis, and at telomeres as well as from aborted topoisomerase reactions, collapsed replication forks, and other stressors. Damaged DNA ends are dangerous in cells and if left unrepaired can lead to genomic rearrangement, loss of genetic information, and eventually cancer. Mre11 is part of the Mre11–Rad50–Nbs1 complex that recognizes DNA double-strand breaks and has exonuclease and endonuclease activities that help to initiate the repair processes to resolve these broken DNA ends. In fact, these activities are crucial for proper DNA damage repair pathway choice. Here, using Pyrococcus furiosus Mre11, we question how two Mre11 separation-of-function mutants, one previously described but the second first described here, maintain endonuclease activity in the absence of exonuclease activity. To start, we performed solution-state NMR experiments to assign the side-chain methyl groups of the 64-kDa Mre11 nuclease and capping domains, which allowed us to describe the structural differences between Mre11 bound to exo- and endonuclease substrates. Then, through biochemical and biophysical characterization, including NMR structural and dynamics studies, we compared the two mutants and determined that both affect the dynamic features and double-stranded DNA binding properties of Mre11, but in different ways. In total, our results illuminate the structural and dynamic landscape of Mre11 nuclease function.
KW - DNA double-strand break repair
KW - Mre11–Rad50
KW - NMR
KW - separation of function
KW - side-chain methyl group
UR - http://www.scopus.com/inward/record.url?scp=85083291039&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2020.03.030
DO - 10.1016/j.jmb.2020.03.030
M3 - Article
C2 - 32246962
AN - SCOPUS:85083291039
VL - 432
SP - 3289
EP - 3308
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 10
ER -