Early dissociation of nuclear factor I from the origin during initiation of adenovirus DNA replication studied by origin immobilization

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Abstract
The DNA-bindlng domain of Nuclear Factor I (NFIBD) enhances Initiation of adenovirus DNA replication up to 50-fold by binding to the auxiliary region of the origin and positioning the viral DNA polymerase. To study if and when NFIBD dissociates from the template, we Immobilized origin DNA to glutathlone - agarose beads by means of a GST-NFIBD fusion protein. This Immobilized template is active in replication. By analyzing the release of prelabeled templates from the beads under different conditions, we show that NFIBD dissociates already early during Initiation. During prelnltiation NFIBD remains bound, but as soon as dC-TP, 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 intermediate, we explain our results by conformatlonal changes occurring in the polymerase during initiation leading to disruption of both the interaction between the polymerase and NFI as well as the interaction between NFI and the DNA.