A short carboxyl-terminal tail is required for single-stranded DNA binding, higher-order structural organization, and...

Area of Science:

• Mitochondrial biology
• DNA repair mechanisms
• Protein structure-function relationships

Background:

• Mgm101 is a Rad52-type single-stranded annealing protein (SSAP) essential for mitochondrial DNA (mtDNA) maintenance.
• Mgm101 functions as large oligomeric rings, but the role of its C-terminal tail is uncharacterized.

Purpose of the Study:

• To elucidate the function of the conserved 32-amino acid carboxyl-terminal tail (Mgm101(238-269)) of Mgm101.
• To determine the tail's role in mtDNA maintenance, single-stranded DNA (ssDNA) binding, and protein structure.

Main Methods:

• Site-directed mutagenesis of specific amino acid residues within the Mgm101 C-tail.
• Assessment of mtDNA maintenance and stability under various conditions (37°C, oxidative stress).
• Analysis of ssDNA binding affinity and protein oligomeric state using biochemical assays.
• In vivo protein stability studies.

Main Results:

• Specific residues (Lys-253, Trp-257, Arg-259, Tyr-268) are essential for mtDNA maintenance.
• Mutations affecting Lys-251, Arg-252, Lys-260, and Tyr-266 impact mtDNA stability under stress.
• The Y268A mutation impairs ssDNA binding without affecting ring structure, while W257A and R259A mutations disrupt oligomerization and lead to degradation.
• The C-tail alone mediates ssDNA binding, suggesting its exposed location on the ring.

Conclusions:

• The Mgm101 C-tail is critical for ssDNA binding, structural integrity, and stability, directly impacting mtDNA maintenance.
• The C-tail likely interacts with ssDNA via electrostatic and base-stacking forces, potentially remodeling the Mgm101 ring.
• These findings offer insights into SSAP function in DNA repair and genome maintenance, with potential implications for broader DNA repair pathways.