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. 1996 Jun 1;24(11):2166–2175. doi: 10.1093/nar/24.11.2166

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

K G Belanger 1, C Mirzayan 1, H E Kreuzer 1, B M Alberts 1, K N Kreuzer 1
PMCID: PMC145913  PMID: 8668550

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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