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Terminal protein-primed amplification of heterologous DNA with a minimal replication system based on phage Phi29.

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 ?29 DNA replication machinery and producing DNA with TP covalently attached to the 5' end. The amplification requires four ?29 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 ?29 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 ?29 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.

SUBMITTER: Mencia M

PROVIDER: S-EPMC3219123 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Terminal protein-primed amplification of heterologous DNA with a minimal replication system based on phage Phi29.

Mencía Mario M Mencía Mario M Gella Pablo P Camacho Ana A de Vega Miguel M Salas Margarita M

Proceedings of the National Academy of Sciences of the United States of America 20111107 46

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) ...[more]

PMID: 22065756

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Project description:Bacillus subtilis phage ?29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5' ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the ?29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for ?29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in ?29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the ?29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3'-5' exonucleolysis and pyrophosphorolysis. The resolution of the ?29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made ?29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of ?29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and localization of the TP at the bacterial nucleoid, where viral DNA replication takes place. The biochemical properties of the ?29 DBP and SSB and their function in the initiation and elongation of ?29 DNA replication, respectively, will be described.

Dataset Information

Terminal protein-primed amplification of heterologous DNA with a minimal replication system based on phage Phi29.

Publications

Terminal protein-primed amplification of heterologous DNA with a minimal replication system based on phage Phi29.

Similar Datasets

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