CHARACTERIZATION OF THE HELA-CELL DNA-POLYMERASE ALPHA-ASSOCIATED AP(4)A BINDING-PROTEIN BY PHOTOAFFINITY-LABELING

The ubiquitous dinucleotide diadenosine tetraphosphate (Ap(4)A) has been proposed to be involved in DNA replication and cell proliferation, DNA repair, platelet aggregation, and vascular tonus. A protein binding to Ap(4)A is associated with a multiprotein form of DNA polymerase alpha (pol alpha(2)) in HeLa cells. We have purified the pol alpha-associated Ap(4)A binding protein to homogeneity. The Ap(4)A binding protein is resolved into two polypeptides of 45 and 22 kDa, designated as A(1) and A(2), respectively. We have utilized [alpha-P-32]8-N-3-Ap(4)A to label the purified binding protein, and by cross-linking the photoaffinity label we have determined that Ap(4)A binds to the A(1) subunit. No binding to the ligand is observed with the A(2) subunit. Photoaffinity labeling is saturated with approximately 0.4 mu M photolabel, with a half-maximal binding at 0.15 mu M. The labeling is UV-dependent and is competed by both 8-N-3-Ap(4)A and Ap(4)A. 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 Ap(4)A. Of the diadenosine polyphosphates, Ap(4)A and Ap(5)A competed for binding, while Ap(2)A and Ap(3)A did not compete for binding. Further, the presence of at least one adenosine may be necessary since Ap(4)G competes but Gp(4)G does not compete for binding to the protein. Various methylene bisphosphonate and thiophosphate analogs of Ap(4)A were tested to see their effect on photoaffinity labeling with 8-N-3-Ap(4)A. Significant differences were observed among the various analogs in their ability to prevent the photoaffinity labeling of the ligand to the binding protein.