Diadenosine Tetraphosphate Binding Protein from Human Hela Cells: Purification and Characterization

The ubiquitous dinucleotide P¹ P⁴-di(adenosine-5ʹ) tetraphosphate (Ap₄A) has been proposed to be involved in DNA replication and cell proliferation, DNA repair, platelet aggregation, and vascular tonus. A protein binding specifically to Ap₄A is associated with a multiprotein form of DNA polymerase a (pol a₂) in HeLa cells. The Ap₄A binding protein from HeLa cells has been purified to homogeneity starting from pol a₂ complex. The Ap₄A binding protein is hydrophobic and is resolved from the pol a₂ complex by hydrophobic interaction chromatography on butyl-Sepharose and subsequently purified to homogeneity by chromatography on Mono-Q and Superose-12 FPLC columns. The Ap₄A binding activity elutes as a single symmetrical peak upon gel filtration with a molecular mass of 200 kDa. Upon polyacrylamide gel electrophoresis under nondenaturing conditions, the purified protein migrates as a single protein of 200 kDa. Upon electrophoresis under denaturing conditions, the binding activity is resolved into two polypeptides of 45 and 22 kDa, designated as A! and A₂, respectively. A, and A₂ can be cross-linked using the homobifunctional cross-linking agent disuccinimidyl suberate. The cross-linked protein migrates as a single protein of 210 kDa on polyacrylamide gels under denaturing conditions, suggesting that these two polypeptides are subunits of a single protein. The purified protein binds Ap₄A efficiently, and by Scatchard analysis, we have determined a dissociation constant of 0.2uM, indicating high affinity of Ap₄A binding protein to its ligand. ATP is not required for the binding activity. The nonionic detergent Triton X-100 is necessary for stabilizing the purified protein. Amino acid composition analysis indicates that A, and A₂ are distinct.