The mutation R107Q alters mtSSB ssDNA compaction ability and binding dynamics

Area of Science:

• Mitochondrial biology
• Molecular genetics
• Biochemistry

Background:

• Mitochondrial single-stranded DNA-binding protein (mtSSB) is crucial for mitochondrial DNA (mtDNA) replication.
• mtSSB variants are implicated in autosomal dominant mitochondrial optic atrophy and retinal dystrophy.

Purpose of the Study:

• To investigate the molecular mechanisms underlying the severe R107Q mtSSB variant.
• To elucidate the functional consequences of the R107Q mutation on mtSSB's interaction with single-stranded DNA (ssDNA).

Main Methods:

• In vitro analysis of mtSSB R107Q oligomeric state.
• Single-molecule approaches to assess ssDNA binding and compaction.
• Real-time competition assays for ssDNA binding.
• Molecular modeling of ssDNA interaction with mtSSB.

Main Results:

• The mtSSBR107Q mutant forms stable tetramers in vitro.
• mtSSBR107Q exhibits reduced ssDNA compaction and increased ssDNA dissociation compared to wild-type (WT) mtSSB.
• WT mtSSB demonstrated a significant advantage over mtSSBR107Q in ssDNA binding competition.
• Molecular modeling suggests the R107Q mutation disrupts electrostatic interactions, reducing ssDNA binding sites.

Conclusions:

• The R107Q mutation severely impairs mtSSB's ssDNA-binding and compaction capabilities.
• These functional deficits are likely due to weakened ssDNA wrapping efficiency and altered protein-ssDNA interactions.
• The findings provide molecular insights into the pathogenesis of mtSSB-associated mitochondrial diseases.