MITOCHONDRIAL SINGLE-STRANDED DNA-BINDING PROTEIN FROM DROSOPHILA EMBRYOS - PHYSICAL AND BIOCHEMICAL-CHARACTERIZATION

Using a stringent purification procedure on single-stranded DNA cellul ose, we have isolated the mitochondrial single-stranded DNA-binding pr otein from Drosophila melanogaster embryos. Its identity is demonstrat ed by amino-terminal sequencing of the homogeneous protein and by its localization to a mitochondrial protein fraction. The mitochondrial pr otein is immunologically and biochemically distinct from the previousl y characterized nuclear replication protein A from Drosophila (Mitsis, P. G., Kowalczykowski, S. C., and Lehman, I. R. (1993) Biochemistry 3 2, 5257-5266; Marton, R. F., Thommes, P., and Cotterill, S. (1994) FEE S Lett. 342, 139-144). It consists of a single polypeptide of 18 kDa, which is responsible for the DNA binding activity. Sedimentation analy sis suggests that D. melanogaster mitochondrial single stranded DNA-bi nding protein exists as a homo-oligomer, possibly a tetramer, in solut ion. The protein binds to DNA in its single-stranded form with a stron g preference over double-stranded DNA or RNG and binds to polypyrimidi nes preferentially over polypurines. Drosophila mitochondrial single-s tranded DNA-binding protein exhibits a greater affinity for long oligo nucleotides as compared to short ones, yet does not show high cooperat ivity. Its binding site size, determined by competition studies and by fluorescence quenching, is approximately 17 nucleotides under low sal t conditions, and increases in the presence of greater than 150 mM NaC l. The homogeneous protein stimulates the activity of mitochondrial DN A polymerase from D. melanogaster embryos, increasing dramatically the rate of initiation of DNA synthesis on a singly primed DNA template.