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...

Replication protein A (RP-A) is an essential single-stranded DNA binding protein (SSB) involved in the initiation and elongation phases of eukaryotic DNA replication. It has the ability to bind single-stranded DNA extremely tightly and possesses a characteristic hetero-trimeric structure. Here we present a method for the purification of RP-A from Drosophila melanogasfer embryos. Drosophila RP-A (dRP-A) has subunits of about 66, 31 and 8 kDa, in line with analogues from other species. It binds single-stranded DNA very tightly via the large subunit. The complete protein has at least a lO-to 20-fold preference for singe-stranded DNA over double-s~anded DNA and it appears that binding is only weakly co-operative. Band shift experiments suggest that it has an approx~ate site covering the size of 16 nucleotides or less, however, it shows a greater affinity for long oligonucleotides than for short ones. We also demonsttate that dRP-A can stimulate the activity of its homologous DNA polymerase a in excess of 20 fold. Analysis of the protein's abundance during embryo development indicates that it varies in a manner akin to other replication proteins.

We have purified a DNA helicase (dhel l) from early Drosophila embryos. dhel l co-purifies with the single-stranded DNA binding protein dRP-A over two purification steps, however, the proteins can be separated by their different native molecular weight, with dhel l activity co-sedimenting with a polypeptide of approximately 200 kDa and a sedimentation coefficient of 8.6 S. The enzyme needs ATP hydrolysis and divalent cations for displacement activity. It is very salt sensitive, having a Mg2+ optimum of 0.5 mM and being inhibited by NaCl concentration > 10 mM. Dhel l moves 5'-->3' on the DNA strand to which it is bound. Unwinding activity decreases with increasing length of the double-stranded region suggesting a distributive mode of action. However, addition of dRP-A to the displacement reaction stimulates the activity on substrates with >300 nucleotides double-stranded region suggesting a specific interaction between these two proteins.

We have purified a DNA helicase from Drosophila embryos by following unwinding activity during the purification of the cellular single-stranded DNA-binding protein dRP-A. This DNA helicase unwinds DNA 5' to 3', has a salt-tolerant activity, and has a preference for purine triphosphates as cofactors for the unwinding reaction. The purified enzyme consists of a single polypeptide of 120 kDa, which cosediments with the helicase activity. Sedimentation analysis suggests that this polypeptide exists as a monomer under high and low salt conditions. Dhel 11 is able to unwind long stretches of DNA, but with decreased efficiency. Addition of Escherichia coli-like single-stranded DNA-binding proteins stimulates the unwinding activity at least 1 0-fold on substrates greater than 200 nucleotides. In particular, the mitochondria1 single-stranded DNA-binding protein isolated from Ilrosophila embryos is able to stimulate unwinding by dhel 11. These properties show that the helicase described is different from another Drosophilu helicase dhel I ; it has thus has been classified as dhel 11.