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Tetramerization and single-stranded DNA binding properties of native and mutated forms of murine mitochondrial
1Departments of Internal Medicine and Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-8573, USA.
Abstract:
We examined previously unexplored aspects of the tetramerization and single-stranded DNA (ssDNA) binding properties of native, precursor, and mutated forms of mitochondrial ssDNA-binding protein (mtSSB) from a mammalian organism (mouse). Tetramic forms of mtSSB reassemble spontaneously after thermal denaturation and undergo subunit exchange. Binding of mtSSB to ssDNA as a function of protein concentration is nonlinear, suggesting a concentration-dependent transition in intrinsic binding affinity and in the topology of the DNA-protein complex. The cleavable presequence at the amino terminus of the precursor form of mtSSB does not disrupt tetramer formation but has a specific inhibitory effect on DNA binding that is not seen in a fusion protein that substitutes a bulkier peptide moiety in this position. Mutated forms of mtSSB bearing amino acid substitutions at highly conserved amino acid positions exhibit subtle or severe defects in ssDNA binding activity and/or tetramerization, even when assembled into heterotetramers in combination with wild-type mtSSB monomers. These experiments provide new insights into structural and functional properties of mammalian mtSSB and have implications for the pathogenesis of human diseases resulting from defects in mtDNA replication.
Insights
Mammalian mitochondrial single-stranded DNA-binding protein (mtSSB) tetramers reassemble and exchange subunits. Protein concentration influences mtSSB
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Mitochondrial single-stranded DNA-binding protein (mtSSB) is crucial for maintaining mitochondrial DNA (mtDNA) stability and replication.
- Understanding mtSSB's structural and functional properties is essential for comprehending mtDNA maintenance and associated human diseases.
Purpose of the Study:
- To investigate the tetramerization and single-stranded DNA (ssDNA) binding characteristics of mouse mtSSB.
- To analyze the impact of the precursor's N-terminal presequence and specific mutations on mtSSB function.
Main Methods:
- Thermal denaturation and reassembly assays to study tetramerization.
- Electrophoretic mobility shift assays (EMSAs) to assess ssDNA binding affinity.
- Site-directed mutagenesis to create and analyze mutant mtSSB forms.
Main Results:
- Mammalian mtSSB tetramers spontaneously reassemble after denaturation and exhibit subunit exchange.
- mtSSB binding to ssDNA is concentration-dependent, indicating altered affinity and complex topology.
- The precursor's cleavable presequence inhibits DNA binding, unlike a bulkier peptide substitution.
- Mutant mtSSB forms show defects in ssDNA binding and/or tetramerization, even in heterotetramers.
Conclusions:
- Mammalian mtSSB possesses unique tetramerization and ssDNA binding properties.
- The N-terminal presequence and conserved residues play critical roles in mtSSB function.
- Dysfunctional mtSSB may contribute to human diseases linked to mtDNA replication defects.