Mitochondrial Single-stranded DNA-binding Protein from Drosophila Embryos

Abstract

Using a stringent purification procedure on singlestranded DNA cellulose, we have isolated the mitochondrial single-stranded DNA-binding protein from Drosophila melanogaster embryos. Its identity is demonstrated by amino-terminal sequencing of the homogeneous protein and by its localization to a mitochondrial protein fraction. The mitochondrial protein is immunologically and biochemically distinct from the previously characterized nuclear replication protein A from Drosophila (Mitsis, P. G., Kowalczykowski, S. C., and Lehman, I. R. Many of the processes involved in DNA metabolism including DNA replication, recombination, and repair, generate intermediates containing single-stranded regions of DNA. These regions are stabilized and kept accessible for the various catalytic processes by the binding of single-stranded DNA-binding proteins (SSBs).1 Prokaryotic SSBs (e.g. Escherichia coli (Eco) SSB and bacteriophage T4 gene 32 protein) are generally small proteins which bind to single-stranded DNA (ssDNA) with high affinity. They show high specificity for ssDNA over doublestranded DNA (dsDNA) and RNA, but display little sequence specificity (reviewed in Refs. 1-3). Although they do not exhibit direct catalytic function, they stimulate DNA replication in vitro. Mitochondrial DNA replication is independent from chromosomal DNA replication and is carried out largely with specific mitochondrial replication proteins including the mitochondrial DNA polymerase (pol ␥) and an SSB (mtSSB) distinct from the nuclear SSB, replication protein A (RP-A). mtSSB appears to serve an important function during mtDNA replication, by stabilizing the displaced ssDNA that is the template for lagging DNA strand synthesis (4). mtSSBs have been isolated from several species including rat (4, 5), Xenopus laevis (6), and yeast (7). These proteins consist of a single small (13-16 kDa) polypeptide, which shows a high degree of similarity to Eco SSB in its primary structure (7,8). Although all the functions of mtSSB in mtDNA metabolism have not been defined, it is critical for replication, because deletion of the yeast protein (RIM1) causes loss of mitochondrial DNA (7). Consistent with a role in mtDNA replication, interactions between mtSSB and other mitochondrial replication proteins have been observed. In vitro studies indicate that under some conditions, the rat and X. laevis mtSSBs stimulate partially purified forms of mitochondrial DNA polymerase (9, 10), and a putative human mtSSB stimulates human pol ␥ (11). In addition, genetic evidence from yeast suggests an interaction between RIM1 and the mtDNA helicase, PIF1 (7).We have purified a single-stranded DNA-binding protein from Drosophila embryos (hereafter called Dm mtSSB) to near homogeneity. Its physical and biochemical properties demonstrate that it is distinct from the nuclear SSB, dRP-A, but has a high degree of similarity to Eco SSB and to eukaryotic mtSSBs. Further, its functional interaction with the near-homogeneous mitochondrial DNA polymerase from Drosophila melanogaster embryos (1...

Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

“…We would suggest that this derives from a different mechanism, because both these and the parent deletion mutant ⌬␥3 retain strong subunit interactions that are comparable with wild-type pol ␥. One interesting possibility is that the reduced processivity of the L558A and F578A mutants is related to defective interaction with mtSSB, a factor that increases strongly both the activity and processivity of wild-type DNA pol ␥ (37,39). Notably, the stimulation of DNA polymerase activity on M13 DNA by mtSSB is reduced 4 -5-fold in the L558A and F578A mutant holoenzymes as compared with wild type (6 -7-versus 30-fold stimulation).…”

“…Mutants and mtSSB-Mitochondrial single-stranded DNAbinding protein stimulates initiation and elongation of DNA strands by wild-type pol ␥ and increases pol ␥ processivity (29,37). Functional interaction between spacer region mutants and mtSSB were evaluated by assaying DNA polymerase activity in the presence or absence of mtSSB using oligonucleotide-primed FIG.…”

Physiological and Biochemical Defects in Functional Interactions of Mitochondrial DNA Polymerase and DNA-binding Mutants of Single-stranded DNA-binding Protein

“…Addition of mtSSB to the reaction lowers the KCl optimum ϳ8-fold to 15-30 mM, while stimulating activity at low salt up to 18-fold (2,5). Surprisingly, although the DNA-binding activities of the mutants with single amino acid substitutions were all within 10% of that of the wild-type mtSSB at 30 mM KCl, defects in their abilities to stimulate DNA synthesis by pol ␥ were apparent in the range of 0 -30 mM KCl (Fig.…”

“…previous studies documented a functional interaction between pol ␥ and mtSSB in replicating single-stranded DNAs that mimic lagging DNA strand synthesis during mtDNA replication (2,5). We sought to explore the possible consequences of perturbing that interaction, both in biochemical assays and in vivo.…”

“…The standard assay for stimulation of pol ␥ by mtSSB on single-stranded DNA substrates is performed at 30 mM KCl, where maximal DNA synthesis combined with the highest enzyme processivity is observed (4,5). Under this ionic condition, all of the mtSSB variants with single amino acid substitutions displayed DNA-binding activities Ն 90% of wild type, whereas the double mutant W79T/F85A showed 50% of wild-type DNA binding (Fig.…”