Formation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H.

Abstract

A plasmid that consists of an 812-base-pair segment containing the replication origin of lasmid ColEl and of a 1240-base-pair segment containing a' lactamase gene has been constructed. The plasmid DNA ias three principal sites where transcription is initiated in vitro. One is located in the CoIEl segment 555 nucleotides upstream from the origin. Most transcription from this site extends past the origin; some of the transcripts form hybrids spontaneously with the template at their 3' portions. Cleavage of these transcripts by RNase H generates 3' termini at the origin region. When DNA polymerase I is included in the reaction along with RNA polymerase and RNase H, dAMP or dCMP is added directly onto the cleaved RNA molecules, most of which retain the intact 5' terminus. The addition of a deoxyribonucleotide to the cleaved RNA can be regarded as the first step of ColEl DNA synthesis. Once it has served as a primer, the RNA is eliminated from the product by RNase H.Closed circular DNA of plasmid ColEl of Escherichia coil can be replicated in vitro by a soluble enzyme system (1). Replication starts most frequently at any of the three consecutive nucleotides (dA, dA, and dG) located at a unique position on the DNA (2). No plasmid-encoded protein is required for this replication (3). Initiation of replication can be demonstrated with a mixture of three highly purified E. coil enzymes: RNA polymerase, DNA polymerase I, and RNase H (4), an enzyme that creates a 3'-OH end by endonucleolytic cleavage of RNA that is hybridized to DNA (5). Previously we proposed (4) that RNA polymerase synthesizes a transcript that is processed by RNase H and then used as a primer by DNA polymerase L. Here we report the identification of such a transcript and demonstrate that it is processed by RNase H to form the primer. The replication of ColEl has been recently reviewed (6-8).MATERIALS AND METHODS Plasmid DNA. Plasmid pNT7 which contains the replication origin of ColEl and the f3-lactamase gene of the Tn3 transposon was derived from pNT5 (9) and pBR322 (10) as described below. Supercoiled molecules of plasmid DNA were purified as described (4).Enzymes. E. coli RNA polymerase (600 units/mg) was the holoenzyme (4, 11). DNA polymerase 1 (15,000 units/mg) (12) was a gift of A. Kornberg. RNase H (470,000 units/mg) was prepared as described (13) with the following modifications: The first DEAE-cellulose chromatography was performed at pH 8.9 instead of pH 7.5. An additional DEAE-cellulose chromatography was performed at pH 7.5 without a salt gradient. Sephadex G-50 chromatography replaced glycerol gradient centrifugation. NaDodSO4/polyacrylamide gel electrophoresis (14) A linear gradient gel of 2-20% polyacrylamide was also used. The 25% gels were 0.4 mm thick, whereas the others were 1.5 mm thick. The length of the slab gels was 38 cm and their width was 16 or 33 cm. Samples dissolved in 1 mM EDTA/5 M urea were heated at 80'C for 3 min immediately before loading. To recover nucleic acids from the gel, the gel was crushed and shaken f...

Initiation of simian virus 40 DNA synthesis in vitro.

“…However, nascent DNA preferentially hybridized to clone C Lag (Fig. 3A) (60) genomes as well as analyses of ColEl (24,33) and oriC replication in vivo (25). In light of these reports and the experiments presented here, it is possible that eukaryotic and prokaryotic initiation mechanisms are evolutionarily related.…”

Functional analysis of the role of the A + T-rich region and upstream flanking sequences in simian virus 40 DNA replication.

“…In addition to these structural similarities to the SV40 origin of replication, the involvement of promoter sequences in procaryotic DNA replication has been well documented (10,20). In these instances, the oligoribonucleotide priming process used to initiate DNA synthesis is directed by specific promoter elements which have been localized to specific sequences.…”

Dissecting the functional role of PriA protein‐catalysed primosome assembly in Escherichia coli DNA replication

“…A key feature of this primosome-dependent DNA replication system is the requirement for the unwinding of the duplex parental DNA template to expose the PAS located on the laggingstrand DNA template and convert it to a single-stranded form. In pBR322, this is accomplished by the sequential action of RNA polymerase, RNase H, and DNA polymerase I (Itoh and Tomizawa 1980). Leading-strand DNA synthesis is initiated with the synthesis of a transcript (RNA II) 555 nucleotides upstream of the origin of DNA replication (the position of the first nascent deoxynucleotide residue).…”