EARLY DISSOCIATION OF NUCLEAR FACTOR-I FROM THE ORIGIN DURING INITIATION OF ADENOVIRUS DNA-REPLICATION STUDIED BY ORIGIN IMMOBILIZATION

The DNA-binding domain of Nuclear Factor I (NFIBD) enhances initiation of adenovirus DNA replication up to 50-fold by binding to the auxilia ry region of the origin and positioning the viral DNA polymerase. To s tudy if and when NFIBD dissociates from the template, we immobilized o rigin DNA to glutathione - agarose beads by means of a GST-NFIBD fusio n protein. This immobilized template is active in replication. By anal yzing the release of prelabeled templates from the beads under differe nt conditions, we show that NFIBD dissociates already early during ini tiation. During preinitiation NFIBD remains bound, but as soon as dC-T P, dATP or dTTP are added, efficient dissociation occurs. A much lower dissociation level was induced by addition of dGTP. Since dCTP, dATP and dTTP are required for formation of a pTP-CAT initiation intermedia te, we explain our results by conformational changes occurring in the polymerase during initiation leading to disruption of both the interac tion between the polymerase and NFI as well as the interaction between NFI and the DNA.