Functional Interactions of Mitochondrial DNA Polymerase and Single-stranded DNA-binding Protein

Abstract

Functional interactions between mitochondrial DNA polymerase (pol ␥) and mitochondrial single-stranded DNA-binding protein (mtSSB) from Drosophila embryos have been evaluated with regard to the overall activity of pol ␥ and in partial reactions involving template-primer binding and initiation and idling in DNA strand synthesis. Both the 5؅ 3 3؅ DNA polymerase and 3؅ 3 5؅ exonuclease in pol ␥ are stimulated 15–20-fold on oligo-nucleotide-primed single-stranded DNA by native and recombinant forms of mtSSB. That the extent of stimu-lation is similar for both enzyme activities over a broad range of KCl concentrations suggests their functional coordination and a similar mechanism of stimulation by mtSSB. At the same time, the high mispair specificity of pol ␥ in exonucleolytic hydrolysis is maintained, indi-cating that enhancement of pol ␥ catalytic efficiency is likely not accompanied by increased nucleotide turn-over. DNase I footprinting of pol ␥⅐DNA complexes and initial rate measurements show that mtSSB enhances primer recognition and binding and stimulates 30-fold the rate of initiation of DNA strands. Dissociation stud-ies show that productive complexes of the native pol ␥ heterodimer with template-primer DNA are formed and remain stable in the absence of replication accessory proteins. Single-stranded DNA-binding proteins (SSBs) 1 serve critical roles in DNA replication, repair and recombination (1). Whereas high affinity DNA binding by SSBs can occur inde-pendently of other proteins, both functional and physical inter-actions between SSBs and a variety of enzymes involved in the above processes have been documented. In particular, interac-tions between SSBs and replicative DNA polymerases have been demonstrated in bacterial, nuclear, and viral systems (1). The near-homogeneous mitochondrial DNA polymerase from Drosophila embryos catalyzes relatively efficient DNA synthe-sis on both predominantly double-and single-stranded DNA templates (2, 3), yet its activity and processivity are greatly affected by reaction conditions (4). Mitochondrial SSBs share similar physical and biochemical properties with Escherichia coli SSB (5–10), with which they exhibit a high degree of amino acid sequence conservation (10 –12). Considering the roles served by E. coli SSB in bacterial replication in helix destabi-lization (13) and in enhancing DNA polymerase processivity (14, 15) and fidelity (16, 17), we purified Drosophila mtSSB and studied its effects in in vitro DNA synthesis by pol ␥, in an assay that mimics lagging DNA strand synthesis in mitochon-drial replication (9). These studies allowed the first demonstra-tion of stimulation by a mtSSB of DNA synthesis by a near-homogeneous pol ␥. Our biochemical data are consistent with an important role for mtSSB in mitochondrial DNA replication that has been documented genetically by the fact that a null mutation in the gene for the yeast homolog (RIM1) results in complete loss of mitochondrial DNA in vivo (7). Furthermore, we found that Drosophila mtSSB stimulates pol ␥ by a mech-anism highly similar to that which we found for E. coli SSB (9, 18). Here we demonstrate a dual role for mtSSB in initiation and elongation of DNA strand synthesis catalyzed by pol ␥, and evaluate for the first time the effects of mtSSB on the mispair-specific 3Ј 3 5Ј exonuclease in pol ␥.