Two-dimensional gel analysis of rolling circle replication in the presence and absence of bacteriophage T4 primase.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Belanger, KG; Mirzayan, C; Kreuzer, HE; Alberts, BM; Kreuzer, KN

Published Date

  • June 1, 1996

Published In

Volume / Issue

  • 24 / 11

Start / End Page

  • 2166 - 2175

PubMed ID

  • 8668550

Pubmed Central ID

  • 8668550

International Standard Serial Number (ISSN)

  • 0305-1048

Digital Object Identifier (DOI)

  • 10.1093/nar/24.11.2166

Language

  • eng

Conference Location

  • England