Primer terminus recognition and highly processive replication by Epstein-Barr virus DNA polymerase

Abstract

The Epstein-Barr virus (EBV) DNA polymerase is essential for viral DNA replication in the lytic phase of the EBV life cycle. It efficiently extends RNA primers on the template DNA, suggesting the possible involvement of the EBV DNA polymerase in synthesizing Okazaki fragments from RNA primers on the lagging strand template. Competition experiments revealed that the EBV DNA polymerase had significantly higher affinity for primer termini hybridized to the template DNA than for the single-stranded DNA template or the single-stranded primer itself. ATP was not required either for primer terminus recognition or for sustainment of polymerization. The stimulation of the enzyme by (NH4)2SO4 was dependent on the template/primers utilized. These observations suggest that the primary and secondary structure of the template/primers are important factors for primer terminus recognition by the EBV DNA polymerase. The enzyme elongated synthetic RNA primer annealed to circular single-stranded M13 DNA coated with Escherichia coli single-stranded DNA-binding protein without dissociation. The processivity of the EBV DNA polymerase was strikingly high (greater than 7200 nucleotides) and the rate of polymerization was 12 nucleotides/s per polymerase molecule. The high processing capacity is a desirable feature in the synthesis of multiple copies of the EBV genome in rolling-circle DNA replication.