Signals in the φ29 DNA-terminal protein template for the initiation of phage φ29 DNA replication

Gutiérrez, Julio; Vinós, Javier; Priéto, Ignacio; Méndez, Enrique; Hermoso, José Miguel; Salas, Margarita

doi:10.1016/0042-6822(86)90209-6

Cited by 31 publications

(29 citation statements)

References 15 publications

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“…As seen in Fig. 3, with the wild-type Nf ori sequence (ori(29)Nf), replication was truncated mainly at TP-(dNMP) 12 , although a faint band corresponding to TP-(dNMP) 11 was also detected (Fig. 3 Left).…”

Section: Resultsmentioning

confidence: 89%

“…As seen in Fig. 3, when the mutated molecule ori(29)Nf T1A was used as template, the replicated product was TP-(dNMP) 11 , indicating that the mutation introduced precluded the recovery of the 3Ј terminal position. Interestingly, with ori(29)Nf T2A, the elongated product corresponded mainly to TP-(dNMP) 12 , although a small amount of TP-(dNMP) 10 was also produced.…”

Section: Resultsmentioning

confidence: 99%

“…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).…”

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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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Section: Resultsmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

mentioning

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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“…Previous results showed that the initiation of replication from templates having Φ29 DNA ends without TP was much less efficient than the initiation performed using the Φ29 TP-DNA as template (16). Notwithstanding, there were no experiments in which a comparison had been made between the Φ29 genome (TP-DNA) and other templates having two Φ29 DNA terminal regions without TP, with respect to in vitro amplification using the Φ29 replication machinery as described (10).…”

Section: Resultsmentioning

confidence: 98%

“…These terminal regions have signals for the cooperative binding of protein p6, which stimulates the initiation of replication likely by opening the two strands of the DNA at the very ends of the genome (14). The heterodimer formed by TP and DNA polymerase (15) is recruited to the origin in a process enhanced by the 5′-linked TP (parental TP) (16). Then, the initiation of replication takes place in which the TP is used as a primer by the DNA polymerase followed by a transition stage when the polymerase synthesizes 10 nucleotides, dissociates from the TP, and switches to the DNA-primed elongation mode of replication until it reaches the end of the genome (17).…”

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confidence: 99%

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

Mencı́a

Gella

Camacho

et al. 2011

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

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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...

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The RGD Sequence in Phage ø29 Terminal Protein Is Required for Interaction with ø29 DNA Polymerase

et al. 1998

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The RGD (Arg-Gly-Asp) motif functions as a recognition site for adhesive proteins responsible for a number of cell-cell interactions. Certain viruses use this sequence as a receptor-binding site by interaction with cellular integrins. To elucidate the role of the RGD sequence of the phi29 terminal protein (TP), seven modified TPs were generated by site-directed mutagenesis. Most of the TP mutants were not efficiently used as primers, leading to a reduction of the TP-dAMP complex formation in the presence of the phi29 TP-DNA template. Moreover, these mutant TPs were poorly deoxyadenylylated by phi29 DNA polymerase in the absence of template. Analysis of primer TP/DNA polymerase complex formation showed that the modified TPs were affected in the formation of the heterodimeric complex. These results indicate that the RGD sequence present in phi29 TP is primarily involved in interaction with the viral DNA polymerase.

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Signals in the φ29 DNA-terminal protein template for the initiation of phage φ29 DNA replication

Cited by 31 publications

References 15 publications

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

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

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

The RGD Sequence in Phage ø29 Terminal Protein Is Required for Interaction with ø29 DNA Polymerase

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