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

Prado, Alicia del; Lázaro, José Portolés; Longás, Elisa; Villar, Laurentino; Vega, Miguel de; Salas, Margarita

doi:10.1074/jbc.m115.682278

Cited by 6 publications

(5 citation statements)

References 37 publications

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“…DNA polymerase mutants were obtained using the QuikChange site-directed mutagenesis kit provided by Stratagene, using as template plasmid pJLPM (a derivative of pT7-4w2) 35 containing the viral gene 2 that encodes the wild-type ϕ29 DNA polymerase, or plasmid pT7-3 that harbours ϕ29 DNA polymerase exonuclease deficient mutant D12A/D66A 24 in the case of the mutants Q180A exo− and Q180E exo− . The presence of the desired mutations, as well as the absence of additional ones was determined by sequencing the entire gene.…”

Section: Methodsmentioning

confidence: 99%

New insights into the coordination between the polymerization and 3′-5′ exonuclease activities in ϕ29 DNA polymerase

Prado

Rodrı́guez

Lázaro

et al. 2019

Sci Rep

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Bacteriophage ϕ29 DNA polymerase has two activities: DNA polymerization and 3′-5′ exonucleolysis governed by catalytic sites present in two structurally distant domains. These domains must work together to allow the correct replication of the template and to prevent the accumulation of errors in the newly synthesized DNA strand. ϕ29 DNA polymerase is endowed with a high processivity and strand displacement capacity together with a high fidelity. Previous studies of its crystallographic structure suggested possible interactions of residues of the exonuclease domain like the Gln180 with the fingers subdomain, or water mediated and direct hydrogen bond by the polar groups of residues Tyr101 and Thr189 that could stabilize DNA binding. To analyse their functional importance for the exonuclease activity of ϕ29 DNA polymerase we engineered mutations to encode amino acid substitutions. Our results confirm that both residues, Tyr101 and Thr189 are involved in the 3′-5′ exonuclease activity and in binding the dsDNA. In addition, Tyr101 is playing a role in processivity and Thr189 is an important determinant in the fidelity of the DNA polymerase. On the other hand, the biochemical characterization of the mutant derivatives of residue Gln180 showed how the mutations introduced enhanced the 3′-5′ exonuclease activity of the enzyme. A potential structural conformation prone to degrade the substrate is discussed.

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Section: Methodsmentioning

confidence: 99%

New insights into the coordination between the polymerization and 3′-5′ exonuclease activities in ϕ29 DNA polymerase

Prado

Rodrı́guez

Lázaro

et al. 2019

Sci Rep

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“…The interchanging of the priming domains of the related Φ29 and Nf TPs, allowed us to conclude that this domain is the one responsible for dictating the internal 3′ nucleotide used as template during initiation, the 2nd and 3rd in Φ29 and Nf DNA, respectively (Longás et al, 2008 ). Recently, we have shown that the aromatic residue Phe230 of the Φ29 TP priming loop is the one responsible for positioning the penultimate nucleotide at the polymerization site to direct insertion of the initial dAMP during the initiation reaction, most probably by interacting with the 3′ terminal base, limiting the internalization of the template strand (see del Prado et al, 2015 ; Figure 6B ). To perform TP-DNA full-length synthesis, the TP-dAMP initiation product translocates backwards one position to recover the template information corresponding to the first 3′-T, the so-called sliding-back mechanism that requires a terminal repetition of 2 bp.…”

Section: Initiation Opposite An Internal Templating Nucleotide: a Smamentioning

confidence: 94%

Section: Initiation Opposite An Internal Templating Nucleotide: a Smamentioning

confidence: 99%

DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication

Salas

Holguera

Redrejo-Rodríguez

et al. 2016

Front. Mol. Biosci.

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Bacillus subtilis phage Φ29 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 Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 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 Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3′–5′ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 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 Φ29 DBP and SSB and their function in the initiation and elongation of Φ29 DNA replication, respectively, will be described.

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“…Single-stranded DNA binding protein and DSB were expressed and purified as described in 86 and 52 , respectively. Phi29 DNA polymerase variants S79G, A80T and S79G/A80T were obtained with the QuickChange site-directed mutagenesis kit (Stratagene), using as template for the mutagenic reactions the plasmid pJLPM, which contains the wild-type p2 gene 87 , and following the manufacturer’s instructions. The presence of the desired mutations, as well as the absence of additional ones, was determined by sequencing the entire gene.…”

Section: Methodsmentioning

confidence: 99%

Darwinian Evolution of Self-Replicating DNA in a Synthetic Protocell

Abil,

Restrepo Sierra,

Stan

et al. 2024

Preprint

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Replication, heredity, and evolution are characteristic of Life. We and others have postulated that the reconstruction of a synthetic living system in the laboratory will be contingent on the development of a genetic self-replicator capable of undergoing Darwinian evolution. Although DNA-based life dominates, the in vitro reconstitution of an evolving DNA self-replicator has remained challenging. We hereby emulate in liposome compartments the principles according to which life propagates information and evolves. Using two different experimental configurations supporting intermittent or semi-continuous evolution (i.e., with or without DNA extraction, PCR, and re-encapsulation), we demonstrate sustainable replication of a linear DNA template - encoding the DNA polymerase and terminal protein from the Phi29 bacteriophage - expressed in the protein synthesis using recombinant elements (PURE) system. The self-replicator can survive across multiple rounds of replication-coupled transcription-translation reactions in liposomes and, within only ten evolution rounds, accumulates mutations conferring a selection advantage. Combined data from next-generation sequencing with reverse engineering of some of the enriched mutations reveal nontrivial and context-dependent effects of the introduced mutations. The present results are foundational to build up genetic complexity in an evolving synthetic cell, as well as to study evolutionary processes in a minimal cell-free system.

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

Cited by 6 publications

References 37 publications

New insights into the coordination between the polymerization and 3′-5′ exonuclease activities in ϕ29 DNA polymerase

New insights into the coordination between the polymerization and 3′-5′ exonuclease activities in ϕ29 DNA polymerase

DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication

Darwinian Evolution of Self-Replicating DNA in a Synthetic Protocell

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