2-DIMENSIONAL GEL ANALYSIS OF ROLLING CIRCLE REPLICATION IN THE PRESENCE AND ABSENCE OF BACTERIOPHAGE-T4 PRIMASE

The rolling circle DNA replication structures generated by the in vitr o phage T4 replication system were analyzed using two-dimensional agar ose gels, Replication structures were generated in the presence or abs ence of T4 primase (gp61), permitting the analysis of replication fork s with either duplex or single-stranded tails, A characteristic are sh ape was visualized when forks with single-stranded tails were cleaved by a restriction enzyme with the help of an oligonucleotide that annea ls to restriction sites in the single-stranded tail, After calibrating the gel system with this well-studied rolling circle replication reac tion, we then analyzed the in vivo replication directed by a T4 replic ation origin cloned within a plasmid, DNA samples were generated from infections with either wild-type or primase-deletion mutant phage, The only replicative are that could be detected in the wild-type sample c orresponded to duplex Y forms, consistent with very efficient lagging strand synthesis, Surprisingly, we obtained evidence for both duplex a nd single-stranded DNA tails in the samples from the primase-deficient infection. We conclude that a relatively inefficient mechanism primes lagging strand DNA synthesis in vivo when gp61 is absent.