Isolation and characterisation of dhel II, a DNA helicase from Drosophila melanogaster embryos stimulated by Escherichia coli-type single-stranded-DNA-binding 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 II 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 10-fold on substrates greater than 200 nucleotides. In particular, the mitochondrial single-stranded DNA-binding protein isolated from Drosophila embryos is able to stimulate unwinding by dhel II. These properties show that the helicase described is different from another Drosophila helicase dhel I; it has thus has been classified as dhel II.

Purification and characterisation of a DNA helicase, dheI I, from Drosophila melanogaster embryos.

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.

Mitochondrial single-stranded DNA-binding protein from Drosophila embryos. Physical and biochemical characterization.

Using a stringent purification procedure on single-stranded 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. (1993) Biochemistry 32, 5257-5266; Marton, R. F., Thömmes, P., and Cotterill, S. (1994) FEBS Lett. 342, 139-144). It consists of a single polypeptide of 18 kDa, which is responsible for the DNA binding activity. Sedimentation analysis suggests that D. melanogaster mitochondrial single-stranded DNA-binding protein exists as a homo-oligomer, possibly a tetramer, in solution. The protein binds to DNA in its single-stranded form with a strong preference over double-stranded DNA or RNA, and binds to polypyrimidines preferentially over polypurines. Drosophila mitochondrial single-stranded DNA-binding protein exhibits a greater affinity for long oligonucleotides as compared to short ones, yet does not show high cooperativity. Its binding site size, determined by competition studies and by fluorescence quenching, is approximately 17 nucleotides under low salt conditions, and increases in the presence of greater than 150 mM NaCl. The homogeneous protein stimulates the activity of mitochondrial DNA polymerase from D. melanogaster embryos, increasing dramatically the rate of initiation of DNA synthesis on a singly primed DNA template.

Purification and characterisation of dRP-A: a single-stranded DNA binding protein from Drosophila melanogaster.

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 melanogaster 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 10- to 20-fold preference for single-stranded DNA over double-stranded DNA and it appears that binding is only weakly co-operative. Band shift experiments suggest that it has an approximate site covering the size of 16 nucleotides or less, however, it shows a greater affinity for long oligonucleotides than for short ones. We also demonstrate that dRP-A can stimulate the activity of its homologous DNA polymerase alpha 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.