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. 1974 Mar;71(3):984–988. doi: 10.1073/pnas.71.3.984

Structure of Nascent ϕX174 Replicative Form: Evidence for Discontinuous DNA Replication*

Shlomo Eisenberg 1, David T Denhardt 1
PMCID: PMC388142  PMID: 4522806

Abstract

ϕX174 RF (replicative form) II DNA, labeled in vivo with [methyl-3H]thymidine, was isolated from Escherichia coli polA (DNA polymerase I-deficient) and polA+ cells during RF replication. [32P]dCMP was incorporated into the gaps present in the RF II DNA with [α-32P]dCTP and T4 DNA polymerase. Sedimentation in alkaline sucrose gradients revealed that much of the incorporated 32P was present in a heterogeneous collection of fragments shorter than unit length. Inclusion of polynucleotide ligase in the gap-filling reaction increased the average size of the 32P-labeled fragments. Gel electrophoresis of the products formed by digestion of the 32P-labeled RF II molecules with the restriction nuclease, endonuclease R, indicated that in the population of RF II molecules gaps could occur anywhere in the genome. Competition-annealing experiments provided evidence that the majority of the label incorporated into gaps was present in the minus strand. RF II molecules isolated from polA+ cells were enriched for gaps in a unique region of the genome in comparison with RF II molecules isolated from polA cells. The presence of multiple gaps in the minus strand implies that it is synthesized by a discontinuous mechanism during ϕX RF replication.

Keywords: DNA polymerase, restriction nuclease, competition hybridization, gap-filling, polA

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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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