Structure of the orgin of DNA replication of bacteriophage fd.

Abstract

An RNA-polymerase-protected DNA fragment of 125 nucleotides from the origin of single-strand to doublestrand replication of bacteriophage fd (ori-DNA) was located on the physical map of the phage genome. A stretch of 187 base pairs of DNA including the ori-DNA was sequenced. This DNA segment contains regions with a highly asymmetric pyrimidine/purine distribution next to regions with 2-fold symmetry that form stable hairpin structures in the viral DNA strand. The filamentous bacteriophages fd, M13, and fi contain a small, circular, single-stranded DNA of about 2 X 106 molecular weight (6400 nucleotides). Conversion of the viral DNA into the double-stranded replicative form as well as the replication of both strands of this DNA molecule are initiated and terminated at the same site on the phage genome (1-3), the origin of DNA replication. Detailed in vitro studies of the initiation reaction with the viral DNA as a template have indicated that the origin serves on this DNA strand as a signal for Escherichia coli RNA polymerase to synthesize a short RNA primer (4, 5). A single-strand specific E. coli DNA binding protein (6) has been implicated as an essential auxiliary protein for specific recognition of the origin structure (7,8). In a previous study (8) we isolated and characterized a 120-nucleotide fragment of the origin region from fd (ori-DNA) as an RNA polymerase binding site that is protected by the enzyme against nuclease attack (8). We now report the nucleotide sequence of the ori-DNA fragment. MATERIALS AND METHODSIsolation of fd [32P]DNA (8), pyrimidine tract analysis (8), separation of DNA fragments (8), and oligonucleotide T11C9-primed DNA synthesis (9) have been described. Restriction nucleases used were Hpa II, Hae HI, HinfI, Alu I, and Hha I prepared as described (10). DNA was cleaved in 10 mM Tris-HCl, pH 8/5 mM MgCl2/ 0.5 mM dithiothreitol/50 mM NaCl/5% glycerol. DNA fragments were separated in slab gels of 6% polyacrylamide with 45 mM Tris-borate, ph 8.3/2.5 mM EDTA as buffer.Synthesis of fd Replicative Form (RF) DNA In Vitro. fd DNA (2 nmol) and oligonucleotides from fd RF DNA (chainlength, 10-30; about 10 nmol) were annealed at 450 for 10 min in 10 ml of 0.1 M Tris-HCl/200 mM KCI (11). The mixture was cooled to 200 and the volume was increased to 20 ml with the addition of the four dNTPs (one radiolabeled) to 300,uM each, ATP to 50 MM, MgCl2 to 7 mM, E. coli DNA polymerase I to 40 units/ml, and T4 DNA ligase to 1 unit/ml. DNA synthesis at 200 was followed by determining acid-insoluble radioactivity in small aliquots. Formation of covalently closed fd RF DNA was assessed by the fraction of labeled DNA molecules that was not denatured at pH 12.2 and not retained on nitrocellulose filters (see below). When the latter assay indicated 70-80% ofThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.closed fd RF DNA (2-4 hr), th...

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