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Improvement of ?29 DNA polymerase amplification performance by fusion of DNA binding motifs.

ABSTRACT: Bacteriophage ?29 DNA polymerase is a unique enzyme endowed with two distinctive properties, high processivity and faithful polymerization coupled to strand displacement, that have led to the development of protocols to achieve isothermal amplification of limiting amounts of both circular plasmids and genomic DNA. To enhance the amplification efficiency of ?29 DNA polymerase, we have constructed chimerical DNA polymerases by fusing DNA binding domains to the C terminus of the polymerase. The results show that the addition of Helix-hairpin-Helix [(HhH)(2)] domains increases DNA binding of the hybrid polymerases without hindering their replication rate. In addition, the chimerical DNA polymerases display an improved and faithful multiply primed DNA amplification proficiency on both circular plasmids and genomic DNA and are unique ?29 DNA polymerase variants with enhanced amplification performance. The reported chimerical DNA polymerases will contribute to make ?29 DNA polymerase-based amplification technologies one of the most powerful tools for genomics.

SUBMITTER: de Vega M

PROVIDER: S-EPMC2944734 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs.

de Vega Miguel M Lázaro José M JM Mencía Mario M Mencía Mario M Blanco Luis L Salas Margarita M

Proceedings of the National Academy of Sciences of the United States of America 20100907 38

Bacteriophage ϕ29 DNA polymerase is a unique enzyme endowed with two distinctive properties, high processivity and faithful polymerization coupled to strand displacement, that have led to the development of protocols to achieve isothermal amplification of limiting amounts of both circular plasmids and genomic DNA. To enhance the amplification efficiency of ϕ29 DNA polymerase, we have constructed chimerical DNA polymerases by fusing DNA binding domains to the C terminus of the polymerase. The res ...[more]

PMID: 20823261

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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.

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Improvement of ?29 DNA polymerase amplification performance by fusion of DNA binding motifs.

Publications

Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs.

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