Tetramerization and single-stranded DNA binding properties of native and mutated forms of murine mitochondrial single-stranded DNA-binding proteins.
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.
Li
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K
Departments of Internal Medicine and Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-8573, USA.
Williams
R S
RS
D000595:Amino Acid Sequence; D000818:Animals; D002499:Centrifugation, Density Gradient; D018076:DNA, Complementary / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D014981:Xenopus; D029867:Xenopus Proteins
D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; C484072:mtSSB protein, mouse; C070332:mtSSB-1 protein, Xenopus