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      Formation of a DNA loop at the replication fork generated by bacteriophage T7 replication proteins.

      The Journal of Biological Chemistry

      DNA, Single-Stranded, genetics, Bacteriophage T7, Base Sequence, Biotinylation, DNA, DNA Helicases, metabolism, ultrastructure, DNA Primase, DNA Primers, DNA Replication, DNA, Circular, Bacteriophage M13, DNA, Viral, DNA-Binding Proteins, DNA-Directed DNA Polymerase, Deoxycytosine Nucleotides, chemistry, Molecular Sequence Data, Protein Binding, Viral Proteins

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          Abstract

          Intermediates in the replication of circular and linear M13 double-stranded DNA by bacteriophage T7 proteins have been examined by electron microscopy. Synthesis generated double-stranded DNA molecules containing a single replication fork with a linear duplex tail. A complex presumably consisting of T7 DNA polymerase and gene 4 helicase/primase molecules was present at the fork together with a variable amount of single-stranded DNA sequestered by gene 2.5 single-stranded DNA binding protein. Analysis of the length distribution of Okazaki fragments formed at different helicase/primase concentrations was consistent with coupling of leading and lagging strand replication. Fifteen to forty percent of the templates engaged in replication have a DNA loop at the replication fork. The loops are fully double-stranded with an average length of approximately 1 kilobase. Labeling with biotinylated dCTP showed that the loops consist of newly synthesized DNA, and synchronization experiments using a linear template with a G-less cassette demonstrated that the loops are formed by active displacement of the lagging strand. A long standing feature of models for coupled leading/lagging strand replication has been the presence of a DNA loop at the replication fork. This study provides the first direct demonstration of such loops.

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          9478983

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