Mutation of Conserved Mre11 Residues Alter Protein Dynamics to Separate Nuclease Functions
Samiur Rahman1, Mahtab Beikzadeh1, Marella D Canny1
1Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA.
Abstract:
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.
Insights
DNA double-strand breaks trigger repair pathways. Mre11 nuclease mutants were studied to understand how endonuclease activity persists without exonuclease activity, revealing insights into DNA repair mechanisms.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- DNA double-strand breaks (DSBs) are critical DNA lesions arising from various cellular processes and exogenous agents.
- Unrepaired DSBs can lead to genomic instability, mutations, and diseases like cancer.
- The Mre11-Rad50-Nbs1 (MRN) complex plays a central role in DSB recognition and repair initiation, possessing both exonuclease and endonuclease activities.
Purpose of the Study:
- To investigate how specific Mre11 separation-of-function mutants retain endonuclease activity while lacking exonuclease activity.
- To elucidate the structural and dynamic mechanisms underlying Mre11's dual nuclease functions.
Main Methods:
- Solution-state Nuclear Magnetic Resonance (NMR) spectroscopy was employed to assign methyl groups in Mre11 domains.
- Biochemical and biophysical characterization techniques, including NMR, were used to compare wild-type Mre11 with two separation-of-function mutants.
- Studies focused on Mre11's interaction with exo- and endonuclease substrates.
Main Results:
- Structural differences between Mre11 bound to different substrates were identified using NMR.
- Both Mre11 mutants impacted the dynamic properties of the protein.
- Mutants differentially affected the binding of Mre11 to double-stranded DNA.
Conclusions:
- The study illuminates the structural and dynamic basis of Mre11 nuclease function.
- Understanding Mre11's distinct activities is crucial for comprehending DNA repair pathway fidelity.
- These findings contribute to the knowledge of maintaining genomic integrity.
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