Functional roles of the N- and C-terminal regions of the human mitochondrial single-stranded DNA-binding protein.
Biochemical studies of the mitochondrial DNA (mtDNA) replisome demonstrate that the mtDNA polymerase and the mtDNA helicase are stimulated by the mitochondrial single-stranded DNA-binding protein (mtSSB). Unlike Escherichia coli SSB, bacteriophage T7 gp2.5 and bacteriophage T4 gp32, mtSSBs lack a long, negatively charged C-terminal tail. Furthermore, additional residues at the N-terminus (notwithstanding the mitochondrial presequence) are present in the sequence of species across the animal kingdom. We sought to analyze the functional importance of the N- and C-terminal regions of the human mtSSB in the context of mtDNA replication. We produced the mature wild-type human mtSSB and three terminal deletion variants, and examined their physical and biochemical properties. We demonstrate that the recombinant proteins adopt a tetrameric form, and bind single-stranded DNA with similar affinities. They also stimulate similarly the DNA unwinding activity of the human mtDNA helicase (up to 8-fold). Notably, we find that unlike the high level of stimulation that we observed previously in the Drosophila system, stimulation of DNA synthesis catalyzed by human mtDNA polymerase is only moderate, and occurs over a narrow range of salt concentrations. Interestingly, each of the deletion variants of human mtSSB stimulates DNA synthesis at a higher level than the wild-type protein, indicating that the termini modulate negatively functional interactions with the mitochondrial replicase. We discuss our findings in the context of species-specific components of the mtDNA replisome, and in comparison with various prokaryotic DNA replication machineries.
Department of Biochemistry and Molecular Biology, Center for Mitochondrial Science and Medicine, and Graduate Program in Genetics, Michigan State University, East Lansing, Michigan, United States of America.
Marcos T
MT
Oliveira
Laurie S
LS
Kaguni
D000595:Amino Acid Sequence; D000818:Animals; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D004330:Drosophila; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded