Characterization of the HeLa Cell DNA Polymerase α-Associated Ap₄A Binding Protein by Photoaffinity Labeling

The ubiquitous dinucleotide diadenosine tetraphosphate (Ap₄A) has been proposed to be involved in DNA replication and cell proliferation, DNA repair, platelet aggregation, and vascular tonus. A protein binding to Ap₄A is associated with a multiprotein form of DNA polymerase α (pol α₂) in HeLa cells. We have purified the pol α-associated Ap₄A binding protein to homogeneity. The Ap₄A binding protein is resolved into two polypeptides of 45 and 22 kDa, designated as A₁ and A₂, respectively. We have utilized [α-³²P]8-N₃-Ap₄A to label the purified binding protein, and by cross-linking the photoaffinity label we have determined that Ap₄A binds to the A₁ subunit. No binding to the ligand is observed with the A₂ subunit. Photoaffinity labeling is saturated with approximately 0.4 μM photolabel, with a halfmaximal binding at 0.15 μM. The labeling is UV-dependent and is competed by both 8-N₃-Ap₄A and Ap₄A. Photoaffinity labeling is not affected in the presence of dATP and dGTP and is reduced only in the presence of excess of ATP indicating the specificity of the protein for Ap₄A. Of the diadenosine polyphosphates, Ap₄A and Ap₅A competed for binding, while Ap₂A and Ap₃A did not compete for binding. Further, the presence of at least one adenosine may be necessary since Ap₄G competes but Gp₄G does not compete for binding to the protein. Various methylene bisphosphonate and thiophosphate analogs of Ap₄A were tested to see their effect on photoaffinity labeling with 8-N₃-Ap₄A. Significant differences were observed among the various analogs in their ability to prevent the photoaffinity labeling of the ligand to the binding protein.