A single-stranded DNA binding protein from Drosophila melanogaster: characterization of the heterotrimeric protein and its interaction with single-stranded DNA.
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, omega = 10-300, and high apparent affinity, K omega = (0.7-5) x 10(7) 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 alpha. On the basis of these properties, we conclude that D-SSB is the Drosophila cognate of the human and yeast SSB/RP-A proteins.
Department of Biochemistry, Beckman Center, Stanford University School of Medicine, California 94305.
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PG
Mitsis
S C
SC
Kowalczykowski
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IR
Lehman
D000818:Animals; D001665:Binding Sites; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D004257:DNA Polymerase II / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000737:chemistry* / Q000196:embryology; D046911:Macromolecular Substances; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D004257:DNA Polymerase II