Specific Recognition of Parental Terminal Protein by DNA Polymerase for Initiation of Protein-primed DNA Replication

González-Huici, Víctor; Lázaro, José Portolés; Salas, Margarita; Hermoso, José Miguel

doi:10.1074/jbc.m910058199

Cited by 20 publications

(30 citation statements)

References 44 publications

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“…In summary, the results presented in this work strongly suggest that interactions between the amphipatic ␣-helices of 29 primer and parental TP are important for recognition of the 29 origin of replication and that this process, most likely, also involves interactions between the parental TP and the DNA polymerase (39). Confirmation of these results may come from the determination of the three-dimensional structure of the DNA polymerase-TP complex that will elucidate the position of amphipatic ␣-helix of TP in this complex.…”

Section: Discussionmentioning

confidence: 96%

“…Taking into account the results presented in this work, we consider it also possible that the mutations introduced at positions 80 and 82 of the 29 TP may affect the orientation of the downstream located amphipatic ␣-helix underlying the observed failure of interaction between these mutant primer and mutant parental TPs. Recently, evidence has been obtained that, in addition to the primer TP-parental TP interactions, recognition of the origin of replication of 29 DNA also involves interactions between the DNA polymerase and the parental TP (39).…”

Section: Discussionmentioning

confidence: 99%

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The Putative Coiled Coil Domain of the φ29 Terminal Protein Is a Major Determinant Involved in Recognition of the Origin of Replication

Serna-Rico¹,

Illana²,

Salas

et al. 2000

Journal of Biological Chemistry

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The linear double-stranded genome of phage 29 contains a terminal protein (TP) covalently linked at each 5 DNA end, called parental TP. Initiation of 29 DNA replication starts with the recognition of the origins of replication, constituted by the parental TP-containing DNA ends, by a heterodimer containing 29 DNA polymerase and primer TP. It has been argued that origin recognition involves protein-protein interactions between parental and primer TP. Analysis of the TP sequence revealed that the region between amino acids 84 and 118 has a high probability to form an amphipatic ␣-helix that could be involved in the interaction between parental and primer TP. Therefore, this TP region may be important for origin recognition. To test this hypothesis we introduced various mutations in the predicted amphipatic ␣-helix and analyzed the functionality of the corresponding purified TP mutants. The results obtained show that the identified putative amphipatic ␣-helix of TP is an important determinant involved in origin recognition.Before DNA polymerase duplicates a DNA strand using its complementary strand as template, DNA replication has to be initiated. This process involves several distinct steps such as recognition of the origins, unwinding of double-strand (ds) 1 DNA, and priming (for reviews, see Refs. 1 and 2). Genomes consisting of a linear dsDNA molecule with a terminal protein (TP) covalently linked to their 5Ј-ends have been found in bacteriophages (e.g. 29, 15, Nf, B103, GA-1, Cp-1, and PRD1), animal viruses (e.g. adenoviruses), plasmids (e.g. S1 and Kalilo), and bacteria (e.g. Streptomyces). In most of these cases, initiation of replication has been shown to occur via a so-called protein-priming mechanism, which has been studied extensively for the Bacillus subtilis phage 29 (for reviews, see Refs. 3 and 4). . The formation of this first TP-dAMP covalent complex is directed by the second nucleotide at the 3Ј-end of the template; then, the TP-dAMP complex slides back one nucleotide to recover the information of the terminal nucleotide (8). Next, the 29 DNA polymerase synthesizes a short elongation product before dissociating from the TP (9). Replication, which starts at both DNA ends, is coupled to strand displacement. This results in the generation of so-called type I replication intermediates consisting of fulllength double-stranded 29 DNA molecules with one or more single-stranded DNA branches of varying lengths. When the two converging DNA polymerases merge, a type I replication intermediate becomes physically separated into two type II replication intermediates. Each of these consists of a fulllength 29 DNA molecule in which a portion of the DNA, starting from one end, is double-stranded and the portion spanning to the other end is single-stranded (10, 11). Continuous elongation by the DNA polymerase completes replication of the parental strand.The 29 DNA polymerase and TP form a stable heterodimer (12). This complex recognizes the origin of replication, probably through protein-protein interactions betwee...

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

The Putative Coiled Coil Domain of the φ29 Terminal Protein Is a Major Determinant Involved in Recognition of the Origin of Replication

Serna-Rico¹,

Illana²,

Salas

et al. 2000

Journal of Biological Chemistry

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“…Once both ϕ29 TP and DNA polymerase (shown in cyan) are synthesized, they form a heterodimer that associates with the bacterial nucleoid through the N-terminal DNA-binding domain of the priming TP (shown in red) (Fig. 5B) and recognizes the replication origins at both DNA ends by means of specific interactions with the parental TP (14,15). It seems likely that the association of the ϕ29 TP-DNA to a specific bacterial compartment also might help stabilize the interaction with the heterodimeric complex.…”

Section: Discussionmentioning

confidence: 99%

Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid

Muñoz‐Espín

Holguera

Ballesteros-Plaza

et al. 2010

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

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The mechanism leading to protein-primed DNA replication has been studied extensively in vitro. However, little is known about the in vivo organization of the proteins involved in this fundamental process. Here we show that the terminal proteins (TPs) of phages ϕ29 and PRD1, infecting the distantly related bacteria Bacillus subtilis and Escherichia coli, respectively, associate with the host bacterial nucleoid independently of other viral-encoded proteins. Analyses of phage ϕ29 revealed that the TP N-terminal domain (residues 1-73) possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association. Importantly, we show that in the absence of the TP N-terminal domain the efficiency of ϕ29 DNA replication is severely affected. Moreover, the TP recruits the phage DNA polymerase to the bacterial nucleoid, and both proteins later are redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent way. These data disclose a key function for the TP in vivo: organizing the early viral DNA replication machinery at the cell nucleoid.Bacillus subtilis | phage ø29 | DNA polymerase | DNA-binding | bacterial cytoskeleton P rotein-primed DNA replication is a mechanism used to initiate DNA synthesis in a variety of prokaryotic and eukaryotic organisms (1). Phages such as ϕ29 (infecting Bacillus subtilis) and PRD1 (infecting Escherichia coli) (1, 2), animal viruses such as adenoviruses (1, 3), bacterial species from the Streptomyces genus (1, 4), and viruses infecting Archaea (5-7) possess replication origins at their linear chromosomes constituted by inverted terminal repetitions with a terminal protein (TP) linked to both 5′ genome ends. TPs also have been reported in linear plasmids isolated from bacteria, yeast, fungi, and higher plants (1) and in animal and plant RNA viruses (1).The development of an in vitro replication system with purified proteins and DNA from the B. subtilis phage ϕ29 laid the foundations for investigating the protein-primed mechanism of DNA replication and makes ϕ29 a paradigm for studying this process (1, 2). A schematic overview of the in vitro ϕ29 DNA replication mechanism is shown in Fig. S1. The ϕ29 genome consists of a 19,285-bp linear dsDNA, with a TP of 31 kDa (the parental TP) covalently linked to each 5′ end. DNA replication starts with the recognition of the TP-containing DNA ends by a heterodimer formed by the ϕ29 DNA polymerase and a free TP molecule (the primer TP) (8). The DNA polymerase then catalyzes the formation of a covalent bond between deoxyAMP and the hydroxyl group Ser 232 of the primer TP. Replication is coupled to strand displacement, and continuous elongation of the DNA polymerase from both DNA ends generates replication intermediates that finally converge in the complete duplication of the parental strands (reviewed in ref.2). Phage ϕ29 DNA transcription is divided into early and late stages (9). Fig. S1 shows a genetic and a transcriptional map. Genes 2 and 3, encoding phage DNA polymerase and TP, respectively, are located i...

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“…It contains a linear, dsDNA, 18,754 bp long (GenBank accession number EU622808), with an 8-bp ITR (3Ј-TTTCATTC) (21,22) and a TP of 31 kDa covalently linked to each 5Ј DNA end (21). Previous in vitro analyses showed that Nf also replicates its genome (TP-DNA) by means of a proteinprimed mechanism (23,24).…”

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confidence: 99%

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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Specific Recognition of Parental Terminal Protein by DNA Polymerase for Initiation of Protein-primed DNA Replication

Cited by 20 publications

References 44 publications

The Putative Coiled Coil Domain of the φ29 Terminal Protein Is a Major Determinant Involved in Recognition of the Origin of Replication

The Putative Coiled Coil Domain of the φ29 Terminal Protein Is a Major Determinant Involved in Recognition of the Origin of Replication

Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid

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

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