Replicative DNA polymerases (DNAPs) move along template DNA in a processive manner. The structural basis of the mechanism of translocation has been better studied in the A-family of polymerases than in the B-family of replicative polymerases. To address this issue, we have determined the X-ray crystal structures of phi29 DNAP, a member of the protein-primed subgroup of the B-family of polymerases, complexed with primer-template DNA in the presence or absence of the incoming nucleoside triphosphate, the pre-and post-translocated states, respectively. Comparison of these structures reveals a mechanism of translocation that appears to be facilitated by the coordinated movement of two conserved tyrosine residues into the insertion site. This differs from the mechanism employed by the A-family polymerases, in which a conserved tyrosine moves into the templating and insertion sites during the translocation step. Polymerases from the two families also interact with downstream single-stranded template DNA in very different ways.
The DNA polymerase from phage phi29 is a B family polymerase that initiates replication using a protein as a primer, attaching the first nucleotide of the phage genome to the hydroxyl of a specific serine of the priming protein. The crystal structure of phi29 DNA polymerase determined at 2.2 A resolution provides explanations for its extraordinary processivity and strand displacement activities. Homology modeling suggests that downstream template DNA passes through a tunnel prior to entering the polymerase active site. This tunnel is too small to accommodate double-stranded DNA and requires the separation of template and nontemplate strands. Members of the B family of DNA polymerases that use protein primers contain two sequence insertions: one forms a domain not previously observed in polymerases, while the second resembles the specificity loop of T7 RNA polymerase. The high processivity of phi29 DNA polymerase may be explained by its topological encirclement of both the downstream template and the upstream duplex DNA.
The absolute requirement for primers in the initiation of DNA synthesis poses a problem for replicating the ends of linear chromosomes. The DNA polymerase of bacteriophage /29 solves this problem by using a serine hydroxyl of terminal protein to prime replication. The 3.0 Å resolution structure shows one domain of terminal protein making no interactions, a second binding the polymerase and a third domain containing the priming serine occupying the same binding cleft in the polymerase as duplex DNA does during elongation. Thus, the progressively elongating DNA duplex product must displace this priming domain. Further, this heterodimer of polymerase and terminal protein cannot accommodate upstream template DNA, thereby explaining its specificity for initiating DNA synthesis only at the ends of the bacteriophage genome. We propose a model for the transition from the initiation to the elongation phases in which the priming domain of terminal protein moves out of the active site as polymerase elongates the primer strand. The model indicates that terminal protein should dissociate from polymerase after the incorporation of approximately six nucleotides.
Recent crystallographic studies of 29 DNA polymerase have provided structural insights into its strand displacement and processivity. A specific insertion named terminal protein region 2 (TPR2), present only in protein-primed DNA polymerases, together with the exonuclease, thumb, and palm subdomains, forms two tori capable of interacting with DNA. To analyze the functional role of this insertion, we constructed a 29 DNA polymerase deletion mutant lacking TPR2 amino acid residues Asp-398 to Glu-420. Biochemical analysis of the mutant DNA polymerase indicates that its DNA-binding capacity is diminished, drastically decreasing its processivity. In addition, removal of the TPR2 insertion abolishes the intrinsic capacity of 29 DNA polymerase to perform strand displacement coupled to DNA synthesis. Therefore, the biochemical results described here directly demonstrate that TPR2 plays a critical role in strand displacement and processivity.protein-primed replication ͉ terminal protein region ͉ helicase-like activity ͉ DNA-binding stability D NA replication is a complex multistep process that involves a wide range of proteins and enzymatic activities (1, 2). DNA synthetic activity is provided by DNA polymerases that add nucleotides to the 3Ј-OH end of a primer strand guided by base pairing with the template strand. Polymerases involved in DNA replication are referred to as replicases to distinguish them from other DNA polymerases whose synthetic activities play a role in processes such as DNA repair or recombination. In most DNA replication systems, replication fork movement along the duplex DNA requires an unwinding activity to separate the strands as replication progresses (1, 2). Generally, such activity is not intrinsic to the replicase but is provided either by monomeric or multimeric enzymes called helicases, which melt the dsDNA in an ATP-dependent fashion. In addition, the intrinsic processivity (number of nucleotides incorporated per single DNA polymerase͞DNA-binding event) of most replicases is not high enough to account for the replication of an entire genome, and therefore processivity factors are also required to hold the DNA replicase on the template strand (1, 2).Bacteriophage 29 DNA polymerase is a protein-primed DNA-dependent replicase belonging to the eukaryotic-type family of DNA polymerases (family B). Other members of this family include polymerases with cellular, bacterial, and viral origins (3). 29 DNA polymerase, like many other replicative polymerases, contains both 5Ј-3Ј synthetic and 3Ј-5Ј degradative activities within a single polypeptide chain. Its intrinsic insertion discrimination of 10 4 to 10 6 (4) is further improved 100-fold (5) through proofreading by the exonuclease domain. An extensive mutational analysis of 29 DNA polymerase served to identify the catalytic residues required for these two activities, as well as those responsible for the stabilization of the primer terminus at the respective active sites; these residues are evolutionarily conserved in most DNA polymerases (reviewed ...
By site‐directed mutagenesis in phi29 DNA polymerase, we have analyzed the functional importance of two evolutionarily conserved residues belonging to the 3′‐5′ exonuclease domain of DNA‐dependent DNA polymerases. In Escherichia coli DNA polymerase I, these residues are Thr358 and Asn420, shown by crystallographic analysis to be directly acting as single‐stranded DNA (ssDNA) ligands at the 3′‐5′ exonuclease active site. On the basis of these structural data, single substitution of the corresponding residues of phi29 DNA polymerase, Thr15 and Asn62, produced enzymes with a very reduced or altered capacity to bind ssDNA. Analysis of the residual 3′‐5′ exonuclease activity of these mutant derivatives on ssDNA substrates allowed us to conclude that these two residues do not play a direct role in the catalysis of the reaction. On the other hand, analysis of the 3′‐5′ exonuclease activity on either matched or mismatched primer/template structures showed a critical role of these two highly conserved residues in exonucleolysis under polymerization conditions, i.e. in the proofreading of DNA polymerization errors, an evolutionary advantage of most DNA‐dependent DNA polymerases. Moreover, in contrast to the dual role in 3′‐5′ exonucleolysis and strand displacement previously observed for phi29 DNA polymerase residues acting as metal ligands, the contribution of residues Thr15 and Asn62 appears to be restricted to the proofreading function, by stabilization of the frayed primer‐terminus at the 3′‐5′ exonuclease active site.
By (6, 7). In this reaction, dATP is selected by base complementarity with the second 3'-nucleotide of the template strand (8). After this initiation step, dissociation of the TP-DNA polymerase heterodimer is likely to occur (transition) to replace the TP-DNA polymerase interactions required for initiation by the DNA polymerase-DNA interactions required for the elongation of the newly created DNA primer. Concomitantly, an asymmetric translocation (sliding back) of only TP-dAMP, but not of the template, followed by addition of a new dAMP residue, allows the recovery of the information corresponding to the first template nucleotide (8). During elongation, 429 DNA polymerase catalyzes highly processive polymerization coupled to strand displacement (9), and, therefore, complete replication of both strands proceeds continuously from each terminal priming event. As the two replication forks move, DNA synthesis is initially coupled to strand displacement of long stretches of single-stranded 429 DNA, producing type I replicative intermediates (see Fig. 1 (10,11). Protein p6 (Mr = 11,873) and protein p5 (Mr = 13,212), obtained from 429-infected B. subtilis cells, were purified as described (12, 13). TP-linked DNA from 429 susl4 (1242)
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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