Abstract
Mgm101 is a Rad52-type recombination protein of bacteriophage origin required for the repair and maintenance of mitochondrial DNA (mtDNA). It forms large oligomeric rings of ∼14-fold symmetry that catalyze the annealing of single-stranded DNAs in vitro. In this study, we investigated the structural elements that contribute to this distinctive higher order structural organization and examined its functional implications. A pair of vicinal cysteines, Cys-216 and Cys-217, was found to be essential for mtDNA maintenance. Mutations to the polar serine, the negatively charged aspartic and glutamic acids, and the hydrophobic amino acid alanine all destabilize mtDNA in vivo. The alanine mutants have an increased propensity of forming macroscopic filaments. In contrast, mutations to aspartic acid drastically destabilize the protein and result in unstructured aggregates with severely reduced DNA binding activity. Interestingly, the serine mutants partially disassemble the Mgm101 rings into smaller oligomers. In the case of the C216S mutant, a moderate increase in DNA binding activity was observed. By using small angle x-ray scattering analysis, we found that Mgm101 forms rings of ∼200 Å diameter in solution, consistent with the structure previously established by transmission electron microscopy. We also found that the C216A/C217A double mutant tends to form broken rings, which likely provide free ends for seeding the growth of the super-stable but functionally defective filaments. Taken together, our data underscore the importance of a delicately maintained ring structure critical for Mgm101 activity. We discuss a potential role of Cys-216 and Cys-217 in regulating Mgm101 function and the repair of damaged mtDNA under stress conditions.
Highlights
The Rad52-type mitochondrial recombination protein Mgm101 forms large oligomeric rings, but its functional implication is poorly understood
Substitution by Alanine Revealed That Cys-216 and Cys-217 Are Critical for Mgm101 Function in Vivo—A salient feature of Mgm101 is the formation of large oligomeric rings of ϳ14-fold symmetry with a molecular mass of ϳ400 kDa
Another characteristic feature is that these proteins all form homo-oligomeric rings of 10 –14-fold symmetry [19, 26, 27]
Summary
The Rad52-type mitochondrial recombination protein Mgm101 forms large oligomeric rings, but its functional implication is poorly understood. Mgm101 is a Rad52-type recombination protein of bacteriophage origin required for the repair and maintenance of mitochondrial DNA (mtDNA). It forms large oligomeric rings of ϳ14-fold symmetry that catalyze the annealing of singlestranded DNAs in vitro. Rad or RecA that forms the helical nucleoprotein filaments and initiates strand invasion and homologous pairing within duplex DNA templates In this respect, Rad functions as a recombination mediator for Rad. When bound to ssDNA, Mgm101 forms condensed nucleoprotein filaments like Sak from the lactococcal phage ul36 These observations suggest that Mgm101 may functionally operate like the bacteriophage proteins and catalyze recombination by the single strand annealing mode. We will discuss the possibility that potential modifications of the specific cysteine residues may be important for activating mtDNA transactions in response to oxidative damage or other stresses in mitochondria
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