Functional characterization of highly processive protein-primed DNA polymerases from phages Nf and GA-1, endowed with a potent strand displacement capacity

Longás, Elisa; Vega, Miguel de; Lázaro, José Portolés; Salas, Margarita

doi:10.1093/nar/gkl769

Cited by 15 publications

(25 citation statements)

References 76 publications

(88 reference statements)

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“…The DNA polymerase catalyzes the covalent linkage of the initial dAMP to the hydroxyl group of Ser-232 of the TP and its further elongation (8). Similarly, TP-DNA replication of bacteriophages PRD1 (31,33), Cp-1 (30), and the 29-related phages GA-1 (22,29) and Nf (22,34) was also shown to occur by a protein-primed mechanism, involving its corresponding DNA polymerase and TP. Initiation of 29 DNA replication occurs at the second 3Ј nucleotide of the template.…”

Section: Discussionmentioning

confidence: 99%

“…The results presented in this paper indicate that although the parental TP is not playing a significant role in the recognition of the template nucleotide used to direct the initiation reaction in 29 TP-DNA replication, the correct positioning of the 3Ј terminus of the template strand at the polymerase active site relies on residue Phe-230 of the TP priming loop, allowing the penultimate T to direct the insertion of the initiator dAMP. Site-directed Mutagenesis of 29 TP-TP mutants were obtained using the QuikChange site-directed mutagenesis kit provided by Stratagene, using as template the plasmid pT7-3 that contains the viral gene 3 coding for the TP (22). The presence of the desired mutations, as well as the absence of additional ones was determined by sequencing the entire gene.…”

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confidence: 99%

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

Prado

Lázaro

Longás

et al. 2015

Journal of Biological Chemistry

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Background:The initiation of replication in 29 mainly occurs opposite the 3Ј penultimate nucleotide of the template. Results: Mutations in the TP residue Phe-230 changed the specificity for the templating nucleotide. Conclusion:The results suggest a role for Phe-230 in determining the templating nucleotide. Significance: The results shed light on the positioning of the template nucleotide in organisms that initiate with proteinpriming mechanism.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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

Prado

Lázaro

Longás

et al. 2015

Journal of Biological Chemistry

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“…Wild-type 29 and Nf TPs were expressed in E. coli BL21(DE3) cells harboring the gene cloned into plasmid pT7-3 and further purified as described (24,32). 29 DNA polymerase mutant (D12A/D66A) and Nf DNA polymerase mutant D66A, both exonuclease deficient, were purified as described in ref.…”

Section: Methodsmentioning

confidence: 99%

“…This mutation specifically inactivated the 3Ј to 5Ј exonuclease activity of the enzyme. The expression and purification of the protein was carried out as described for the wild-type Nf DNA polymerase (24).…”

Section: Methodsmentioning

confidence: 99%

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Phage φ29 and Nf terminal protein-priming domain specifies the internal template nucleotide to initiate DNA replication

Longás¹,

Villar²,

Lázaro³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Bacteriophages 29 and Nf from Bacillus subtilis start replication of their linear genome at both DNA ends by a protein-primed mechanism, by which the DNA polymerase, in a template-instructed reaction, adds 5 -dAMP to a molecule of terminal protein (TP) to form the initiation product TP-dAMP. Mutational analysis of the 3 terminal thymines of the Nf DNA end indicated that initiation of Nf DNA replication is directed by the third thymine on the template, the recovery of the 2 terminal nucleotides mainly occurring by a stepwise sliding-back mechanism. By using chimerical TPs, constructed by swapping the priming domain of the related 29 and Nf proteins, we show that this domain is the main structural determinant that dictates the internal 3 nucleotide used as template during initiation.chimerical terminal protein ͉ polymerase ͉ linear genomes ͉ protein-primed replication ͉ sliding-back M any organisms, such as bacteriophages; animal viruses, such as adenovirus, mitochondrial plasmids, linear chromosomes and plasmids of Streptomyces (1); and more recently virus infecting Archaea, such as halovirus (2, 3), possess replication origins constituted by inverted terminal repetitions (ITR) with a terminal protein (TP) linked to both 5Ј ends of their linear chromosomes (4). In these cases, the location of the 2 replication origins allows both strands to be replicated continuously, without requiring asymmetric complexes of DNA polymerase with other accessory proteins to control the different mechanics of continuous and discontinuous DNA synthesis (5). Additionally, the TP provides the OH Ϫ group of a specific serine, threonine or tyrosine to prime initiation of DNA replication from the very ends of the linear chromosome, the TP remaining covalently linked to the 5Ј-DNA ends (parental TP) (1,4,6).The development of an in vitro replication system with highly purified proteins and DNA from bacteriophage 29 of B. subtilis has allowed to lay the foundations of the so-called proteinprimed mechanism of DNA replication (4, 6). 29 has a linear dsDNA, 19,285 bp long, containing a TP of 31 kDa covalently linked to each 5Ј end [TP-DNA (7)] that, together with a 6-bp inverted terminal repeat (3Ј-TTTCAT-5Ј) (8, 9) form part of a minimal replication origin. Once the replication origins are specifically recognized by a heterodimer formed by the DNA polymerase and a free TP molecule (10, 11), the DNA polymerase catalyzes the formation of a covalent bond between dAMP and the hydroxyl group of Ser 232 of the TP, a reaction directed by the second T at the 3Ј end (12). Then, the TP-dAMP initiation product translocates backwards 1 position to recover the template information corresponding to the first T, the so-called sliding-back mechanism, which requires a terminal repetition of 2 bp (12) and provides a way to prevent mutations at the 29 DNA ends during initiation, since the 3Ј-5Ј exonuclease activity of 29 DNA polymerase cannot proofread the TP-linked nucleotide (13).The sliding-back mechanism, occurring also in the 29-related phage GA-1 (14); S...

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Bacteriophage Protein–Protein Interactions

Häuser¹,

Blasche²,

Dokland³

et al. 2012

Advances in Virus Research

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Functional characterization of highly processive protein-primed DNA polymerases from phages Nf and GA-1, endowed with a potent strand displacement capacity

Cited by 15 publications

References 76 publications

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

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

Phage φ29 and Nf terminal protein-priming domain specifies the internal template nucleotide to initiate DNA replication

Bacteriophage Protein–Protein Interactions

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