Terminal protein-primed amplification of heterologous DNA with a minimal replication system based on phage Φ29

Abstract

The DNA amplification performed by terminal protein-primed replication systems has not yet been developed for its general use to produce high amounts of DNA linked to terminal protein (TP). Here we present a method to amplify in vitro heterologous DNAs using the Φ29 DNA replication machinery and producing DNA with TP covalently attached to the 5′ end. The amplification requires four Φ29 proteins, DNA polymerase, TP, single-stranded DNA binding protein and double-stranded DNA binding protein (p6). The DNA to be amplified is inserted between two sequences that are the Φ29 DNA replication origins, consisting of 191 and 194 bp from the left and right ends of the phage genome, respectively. The replication origins do not need to have TP covalently attached beforehand to be functional in amplification and they can be joined to the DNA to be amplified by cloning or ligation. The facts that two functional origins were required at the ends of a linear template DNA and that the kinetics of DNA synthesis was very similar to that obtained using the TP-containing Φ29 genome as template support the proposal that genuine amplification is taking place. Amplification factors of 30-fold have been obtained. Possible applications of DNAs produced by this method are discussed.Φ29 DNA polymerase | origins of replication A lthough the most common systems to start DNA replication are those in which a DNA or an RNA molecule is used as primer, DNA synthesis can also be started using a protein as primer. This type of system initiates DNA synthesis using as an acceptor of the first dNMP, the OH group of a specific serine, threonine, or tyrosine residue of a protein, instead of the 3′ OH group of a ribose or deoxyribose (1). This protein is generally named terminal protein (TP) because it becomes covalently attached at the 5′ termini of the DNA. The TP-primed DNA replication has been studied in a number of systems, such as bacteriophages Φ29, Nf, and GA-1 (2), PRD1 (3), and Cp-1 (4), linear plasmids from bacteria such as pSCL and pSLA2 (5), and in adenovirus (6). In addition, TP-containing DNAs have been identified in linear plasmids of mitochondria, yeast, and plants and in bacterial chromosomes (Streptomyces sp.) (see ref. 1 for a review). From all these, the bacteriophage Φ29 TP-DNA replication system has emerged as the best studied one (7). In vivobased methods to generate linear DNAs with TP attached at the 5′ ends have been described for Streptomyces plasmids (8) and adenovirus (9). These methods take advantage of the observation that a DNA containing the correct terminal sequences but not having TP linked at the ends is capable, after transformation of an appropriate host and selection, to produce inside the cell the corresponding TP-DNA that is stably maintained. However, to date none of these systems has been used to produce TP-DNAs in high amounts appropriate for different molecular biology uses (see below). Regarding the in vitro approaches, as of today, the most efficient system has proved to be the Φ29 DNA replication one. To o...

Functional eukaryotic nuclear localization signals are widespread in terminal proteins of bacteriophages

“…Moreover, using an in vitro TP-primed amplification system that generates DNA molecules containing the TP covalently linked to the 5′ DNA ends (29), we show that the attached Φ29 TP increases gene delivery with respect to that of a control linear DNA, indicating that the attached TP may facilitate the nuclear translocation step. Altogether, our results show that eukaryotic nuclear targeting of TPs of diverse bacteriophages is a widespread feature of these proteins, supporting a possible role in HGT by transferring genes between prokaryotes and eukaryotes.…”

Insights into the Determination of the Templating Nucleotide at the Initiation of φ29 DNA Replication

“…Proteins-TP mutants were expressed in Escherichia coli BL21(DE3) cells harboring gene 3 cloned into plasmid pT7-3 and further purified, essentially as described for the wild-type TP (23). Both, the wild-type 29 DNA polymerase and the exonuclease-deficient mutant D12A/D66A (24) were expressed in E. coli BL21(DE3) cells harboring gene 2 cloned into plasmid pJLPM (a derivative of pT7-4w2 containing the wild-type gene 2 that encodes the 29 DNA polymerase and its ribosomal binding site; the nucleotide T was changed into G in the Shine-Dalgarno sequence) and further purified essentially as described (25).…”

My Life with Bacteriophage φ29

“…The amplification requires the four 29 proteins mentioned above (TP, DNA polymerase, protein p6, and SSB). The DNA to be amplified is inserted between two sequences that are the 29 DNA replication origins, consisting of 191 and 194 bp from the left and right ends of the phage genome, respectively (41). This method provide possibilities regarding amplification of DNA and the generation of hybrid protein-DNA molecules.…”