DISSOCIATION OF THE PROTEIN PRIMER AND DNA-POLYMERASE AFTER INITIATION OF ADENOVIRUS DNA-REPLICATION

Initiation of adenovirus DNA replication occurs by a jumping back mech anism in which the precursor terminal priming protein (pTP) forms a pT P trinucleotide complex (pTP CAT) catalyzed by the viral DNA polymeras e (pol), This covalent complex subsequently jumps back 3 bases to perm it the start of chain elongation, Before initiation, pTP and pol form a tight heterodimer, We investigated the fate of this pTP pol complex during the various steps in replication, Employing in vitro initiation and elongation on both natural viral templates and synthetic oligonuc leotides followed by glycerol gradient separation of the reaction prod ucts, we established that pTP and pol are separated during elongation, Whereas pTP.C and pTP.CA were still bound to the polymerase, after th e formation of pTP.CAT 60% of the pTP pol complex had dissociated, Dis sociation coincides with a change in sensitivity to inhibitors and in K-m for dNTPs, suggesting a conformational change in the polymerase, b oth in the active site and in the pTP interaction domain, In agreement with this, the polymerase becomes a more efficient enzyme after relea se of the pTP primer, We also investigated whether the synthesis of a pTP initiation intermediate is confined to three nucleotides. Employin g synthetic oligonucleotide templates with a sequence repeat of two nu cleotides (GAGAGAGA ... instead of the natural GTAGTA ...) we show tha t G5 rather than G3 is used to start, leading to a pTP tetranucleotide (CTCT) intermediate that subsequently jumps back, This indicates flex ibility in the use of the start site with a preference for the synthes is of three or four nucleotides during initiation rather than two.