title: Two-dimensional gel analysis of rolling circle replication in the presence and absence of bacteriophage T4 primase.
abstract: The rolling circle DNA replication structures generated by the in vitro phage T4 replication system were analyzed using two-dimensional agarose gels. Replication structures were generated in the presence or absence of T4 primase (gp61), permitting the analysis of replication forks with either duplex or single-stranded tails. A characteristic arc shape was visualized when forks with single-stranded tails were cleaved by a restriction enzyme with the help of an oligonucleotide that anneals to restriction sites in the single-stranded tail. After calibrating the gel system with this well-studied rolling circle replication reaction, we then analyzed the in vivo replication directed by a T4 replication origin cloned within a plasmid. DNA samples were generated from infections with either wild-type or primase-deletion mutant phage. The only replicative arc that could be detected in the wild-type sample corresponded to duplex Y forms, consistent with very efficient lagging strand synthesis. Surprisingly, we obtained evidence for both duplex and 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.
authors.affiliation: Department of Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: K G
authors.initials: KG
authors.lastname: Belanger
authors.forename: C
authors.initials: C
authors.lastname: Mirzayan
authors.forename: H E
authors.initials: HE
authors.lastname: Kreuzer
authors.forename: B M
authors.initials: BM
authors.lastname: Alberts
authors.forename: K N
authors.initials: KN
authors.lastname: Kreuzer
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D019915:DNA Primase; D004261:DNA Replication; D004277:DNA, Single-Stranded; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D004277:DNA, Single-Stranded; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; C053688:endodeoxyribonuclease AlwNI; D015252:Deoxyribonucleases, Type II Site-Specific