We describe the purification to near homogeneity of a single-stranded DNA binding protein from 0-18-h embryos of Drosophila melanogaster. Drosophila SSB (D-SSB) is a heterotrimer with subunits of molecular weight of 70 000, 30 000, and 8000. It has a Stokes radius of 48.6 ± 2 A and s20,w = 5.0 ± 0.2 S. The interaction of D-SSB with ssDNA was examined by the quenching of intrinsic protein fluorescence. The binding site size was determined to be n = 22 ± 4 nucleotides with a maximum quenching Qm = 35 ± 3%. Equilibrium titrations indicate that D-SSB binds with low cooperativity, ω = 10-300, and high apparent affinity, Kω = (0.7-5) × 107 M-1 at 225 mM NaCl. Sedimentation of D-SSB bound to small oligonucleotides demonstrates that D-SSB does not require protein association for binding. D-SSB stimulates the extent and processivity of DNA synthesis of its cognate DNA polymerase α. On the basis of these properties, we conclude that D-SSB is the Drosophila cognate of the human and yeast SSB/RP-A proteins.
|Original language||English (US)|
|Number of pages||10|
|State||Published - 1993|
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