Purification Strategies for Drosophila mtDNA Replication Proteins in Native and Recombinant Form: Mitochondrial Single-Stranded DNA-Binding Protein

Single-stranded DNA-binding proteins (SSBs) serve critical roles in DNA replication, repair, and recombination (1 ). Mitochondrial SSBs (mtSSBs) share similar physical and biochemical properties with Escherichia coli SSB (2 –7 ), with which they exhibit a high degree of amino acid sequence conservation (7 –9 ). Considering the roles served by E. coli SSB in bacterial replication in helix destabilization (10 ) and in enhancing DNA polymerase processivity (11 –12 ) and fidelity (13 –14 ), we purified Drosophila mtSSB and studied its effects on in vitro DNA synthesis by pol γ, in an assay that mimics lagging DNA strand synthesis in mitochondrial replication (6 –15 ). Our biochemical data are consistent with an important role for mtSSB in initiation and elongation of DNA strands in mitochondrial DNA (mtDNA) 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 mtDNA in vivo (4 ). More recently, we have demonstrated that an insertion in the third intron of the Drosophila gene (lopo) results in developmental lethality, concomitant with the loss of mtDNA and respiratory capacity (16 ).