The putative coiled coil domain of the phi 29 terminal protein is a major determinant involved in recognition of the origin of replication

The linear double-stranded genome of phage phi29 contains a terminal protein (TP) covalently linked at each 5' DNA end, called parental TP. Initiation of phi29 DNA replication starts with the recognition of the origins of replication, constituted by the parental TP-containing DNA ends, by a heterodimer containing phi29 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 alpha-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 alpha-helix and analyzed the functionality of the corresponding purified TP mutants. The results obtained show that the identified putative amphipatic alpha-helix of TP is an important determinant involved in origin recognition.     

Specific recognition of parental terminal protein by DNA polymerase for initiation of protein-primed DNA replication

The linear genome of Bacillus subtilis phage phi29 has a protein covalently linked to the 5' ends, called parental terminal protein (TP), and is replicated using a free TP as primer. The initiation of phage phi29 DNA replication requires the formation of a DNA polymerase/TP complex that recognizes the replication origins located at the genome ends. The DNA polymerase catalyzes the formation of the initiation complex TP-dAMP, and elongation proceeds coupled to strand displacement. The same mechanism is used by the related phage Nf. However, DNA polymerase and TP from phi29 do not initiate the replication of Nf TP-DNA. To address the question of the specificity of origin recognition, we took advantage of the initiation reaction enhancement in the presence of Mn(2+), allowing us to detect initiation activity in heterologous systems in which DNA polymerase, TP, and template TP-DNA are not from the same phage. Initiation was selectively stimulated when DNA polymerase and TP-DNA were from the same phage, strongly suggesting that specific recognition of origins is brought through an interaction between DNA polymerase and parental TP.     

phi29 DNA polymerase residue Phe128 of the highly conserved (S/T)Lx(2)h motif is required for a stable and functional interaction with the terminal protein

Bacteriophage phi29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as primer for initiation of DNA replication. By multiple sequence alignments of DNA polymerases from such a family, we have been able to identify two amino acid residues specifically conserved in the protein-priming subgroup of DNA polymerases, a phenylalanine contained in the (S/T)Lx(2)h motif, and a glutamate belonging to the Exo III motif. Here, we have studied the functional role of these residues in reactions that are specific for DNA polymerases that use a protein-primed DNA replication mechanism, by site-directed mutagenesis in the corresponding amino acid residues, Phe128 and Glu161 of phi29 DNA polymerase. Mutations introduced at residue Phe128 severely impaired the protein-primed replication capacity of the polymerase, being the interaction with the terminal protein (TP) moderately (mutant F128A) or severely (mutant F128Y) diminished. As a consequence, very few initiation products were obtained, and essentially no transition products were detected. Interestingly, phi29 DNA polymerase mutant F128Y showed a decreased binding affinity for short template DNA molecules. These results, together with the high degree of conservation of Phe128 residue among protein-primed DNA polymerases, suggest a functional role for this amino acid residue in making contacts with the TP during the first steps of genome replication and with DNA in the further replication steps.     

Compartmentalization of phage phi29 DNA replication: interaction between the primer terminal protein and the membrane-associated protein p1

The bacteriophage phi29 replication protein p1 (85 amino acids) is membrane associated in Bacillus subtilis-infected cells. The C-terminal 52 amino acid residues of p1 are sufficient for assembly into protofilament sheet structures. Using chemical cross-linking experiments, we demonstrate here that p1DeltaC43, a C-terminally truncated p1 protein that neither associates with membranes in vivo nor self-interacts in vitro, can interact with the primer terminal protein (TP) in vitro. Like protein p1, plasmid-encoded protein p1DeltaC43 reduces the rate of phi29 DNA replication in vivo in a dosage-dependent manner. We also show that truncated p1 proteins that retain the N-terminal 42 amino acids, when present in excess, interfere with the in vitro formation of the TP.dAMP initiation complex in a reaction that depends on the efficient formation of a primer TP-phi29 DNA polymerase heterodimer. This interference is suppressed by increasing the concentration of either primer TP or phi29 DNA polymerase. We propose a model for initiation of in vivo phi29 DNA replication in which the viral replisome attaches to a membrane-associated p1-based structure.     

Protein-primed DNA replication: a transition between two modes of priming by a unique DNA polymerase.

Phage phi29 from Bacillus subtilis is a paradigm of the protein-primed replication mechanism, in which a single-subunit DNA polymerase is involved in both the specific protein-primed initiation step and normal DNA elongation. To start phi29 DNA replication, the viral DNA polymerase must interact with a free molecule of the viral terminal protein (TP), to prime DNA synthesis once at each phi29 DNA end. The results shown in this paper demonstrate that the DNA polymerase-primer TP heterodimer is not dissociated immediately after initiation. On the contrary, there is a transition stage in which the DNA polymerase synthesizes a five nucleotide-long DNA molecule while complexed with the primer TP, undergoes some structural change during replication of nucleotides 6-9, and finally dissociates from the primer protein when nucleotide 10 is inserted onto the nascent DNA chain. This behaviour probably reflects the polymerase requirement for a DNA primer of a minimum length to efficiently catalyze DNA elongation. The significance of such a limiting transition stage is supported by the finding of abortive replication products consisting of the primer TP linked up to eight nucleotides, detected during in vitro replication of phi29 TP-DNA particularly under conditions that decrease the strand-displacement capacity of phi29 DNA polymerase.     

An aspartic acid residue in TPR-1, a specific region of protein-priming DNA polymerases, is required for the functional interaction with primer terminal protein

A multiple sequence alignment of eukaryotic-type DNA polymerases led to the identification of two regions of amino acid residues that are only present in the group of DNA polymerases that make use of terminal proteins. (TPs) as primers to initiate DNA replication of linear genomes. These amino acid regions (named terminal region (TPR protein-1 and TPR-2) are inserted between the generally conserved motifs Dx(2)SLYP and Kx(3)NSxYG (TPR-1) and motifs Kx(3)NSxYG and YxDTDS (TPR-2) of the eukaryotic-type family of DNA polymerases. We carried out site-directed mutagenesis in two of the most conserved residues of phi29 DNA polymerase TPR-1 to study the possible role of this specific region. Two mutant DNA polymerases, in conserved residues AsP332 and Leu342, were purified and subjected to a detailed biochemical analysis of their enzymatic activities. Both mutant DNA polymerases were essentially normal when assayed for synthetic activities in DNA-primed reactions. However, mutant D332Y was drastically affected in phi29 TP-DNA replication as a consequence of a large reduction in the catalytic efficiency of the protein-primed reactions. The molecular basis of this defect is a non-functional interaction with TP that strongly reduces the activity of the DNA polymerase/TP heterodimer.     

Involvement of phage phi29 DNA polymerase and terminal protein subdomains in conferring specificity during initiation of protein-primed DNA replication

To initiate ϕ29 DNA replication, the DNA polymerase has to form a complex with the homologous primer terminal protein (TP) that further recognizes the replication origins of the homologous TP-DNA placed at both ends of the linear genome. By means of chimerical proteins, constructed by swapping the priming domain of the related ϕ29 and GA-1 TPs, we show that DNA polymerase can form catalytically active heterodimers exclusively with that chimerical TP containing the N-terminal part of the homologous TP, suggesting that the interaction between the polymerase TPR-1 subdomain and the TP N-terminal part is the one mainly responsible for the specificity between both proteins. We also show that the TP N-terminal part assists the proper binding of the priming domain at the polymerase active site. Additionally, a chimerical ϕ29 DNA polymerase containing the GA-1 TPR-1 subdomain could use GA-1 TP, but only in the presence of ϕ29 TP-DNA as template, indicating that parental TP recognition is mainly accomplished by the DNA polymerase. The sequential events occurring during initiation of bacteriophage protein-primed DNA replication are proposed.     

A highly conserved lysine residue in phi29 DNA polymerase is important for correct binding of the templating nucleotide during initiation of phi29 DNA replication

DNA polymerases that initiate replication by protein-priming are able to catalyze terminal protein (TP)-primed initiation, the following transition steps and finally DNA-primed elongation. Therefore, their structures must be able to position sequentially both primers, TP and DNA, at a common binding site. For DNA-templated initiation, these DNA polymerases have to bind the origin of replication as template and TP as primer. It is likely that very precise interactions are required to position both TP and templating nucleotide at the polymerization active site. Such a specificity during TP-priming must rely on specific amino acids that must be evolutionarily conserved in this subfamily of DNA polymerases. By site-directed mutagenesis, we have analyzed the functional significance of Lys392 of phi29 DNA polymerase, immediately adjacent to the Kx3NSxYG motif, and specifically conserved among protein-primed DNA polymerases. During TP-primed initiation, mutations in this residue did not affect untemplated TP-dAMP formation, indicating that the interaction with the initiating nucleotide and TP were not affected, whereas the template-directed initiation activity was severely inhibited. Both mutant DNA polymerases had a wild-type-like (overall) DNA binding activity. We thus infer that residue Lys392 of phi29 DNA polymerase is important for the correct positioning of the templating nucleotide at the polymerization active site, a critical requirement during template-directed TP-priming at phi29 DNA origins. Consequently, mutation of this residue compromised the fidelity of the initiation reaction, not controlled by the 3'-5' exonuclease activity. During DNA-primed polymerization, the mutant polymerases showed a defect in translocation of the template strand. This translocation problem could be the consequence of a more general defect in the stabilization and positioning of a next templating nucleotide at the polymerization active site, during DNA-primed DNA synthesis.     

A conserved insertion in protein-primed DNA polymerases is involved in primer terminus stabilisation

Protein-primed DNA polymerases form a subgroup of the eukaryotic-type DNA polymerases family, also called family B or alpha-like. A multiple amino acid sequence alignment of this subgroup of DNA polymerases led to the identification of two insertions, TPR-1 and TPR-2, in the polymerisation domain. We showed previously that Asp332 of the TPR-1 insertion of phi29 DNA polymerase is involved in the correct orientation of the terminal protein (TP) for the initiation of replication. In this work, the functional role of two other conserved residues from TPR-1, Lys305 and Tyr315, has been analysed. The four mutant derivatives constructed, K305I, K305R, Y315A and Y315F, displayed a wild-type 3'-5' exonuclease activity on single-stranded DNA. However, when assayed on double-stranded DNA such activity was higher than that of the wild-type enzyme. This activity led to a reduced pol/exo ratio, suggesting a defect in stabilising the primer terminus at the polymerase active site. On the other hand, although mutant polymerases K305I and Y315A were able to couple processive DNA polymerisation to strand displacement, they were severely impaired in phi29 TP-DNA replication. The possible role of the TPR-1 insertion in the set of interactions with the nascent chain during the first steps of TP-DNA replication is discussed.     

Involvement of residues of the 29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase

Bacteriophage Φ29 genome consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5' end (TP-DNA) that together with a specific sequence constitutes the replication origins. To initiate replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the origins and initiates replication using as primer the hydroxyl group of TP residue Ser232. The 3D structure of the DNA polymerase/TP heterodimer allowed the identification of TP residues that could be responsible for interaction with the DNA polymerase. Here, we examined the role of TP residues Arg158, Arg169, Glu191, Asp198, Tyr250, Glu252, Gln253 and Arg256 by in vitro analyses of mutant derivatives. The results showed that substitution of these residues had an effect on either the stability of the TP/DNA polymerase complex (R158A) or in the functional interaction of the TP at the polymerization active site (R169A, E191A, Y250A, E252A, Q253A and R256A), affecting the first steps of Φ29 TP-DNA replication. These results allow us to propose a role for these residues in the maintenance of the equilibrium between TP-priming domain stabilization and its gradual exit from the polymerization active site of the DNA polymerase as new DNA is being synthesized.     

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

Bacteriophage φ29 from Bacillus subtilis starts replication of its terminal protein (TP)-DNA by a protein-priming mechanism. To start replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the replication origins, placed at both 5′ ends of the linear chromosome, and initiates replication using as primer the OH-group of Ser-232 of the TP. The initiation of φ29 TP-DNA replication mainly occurs opposite the second nucleotide at the 3′ end of the template. Earlier analyses of the template position that directs the initiation reaction were performed using single-stranded and double-stranded oligonucleotides containing the replication origin sequence without the parental TP. Here, we show that the parental TP has no influence in the determination of the nucleotide used as template in the initiation reaction. Previous studies showed that the priming domain of the primer TP determines the template position used for initiation. The results obtained here using mutant TPs at the priming loop where Ser-232 is located indicate that the aromatic residue Phe-230 is one of the determinants that allows the positioning of the penultimate nucleotide at the polymerization active site to direct insertion of the initiator dAMP during the initiation reaction. The role of Phe-230 in limiting the internalization of the template strand in the polymerization active site is discussed.     

DNA-independent deoxynucleotidylation of the phi 29 terminal protein by the phi 29 DNA polymerase.

In this paper, we show that the phi 29 DNA polymerase, in the absence of DNA, is able to catalyze the formation of a covalent complex between the phi 29 terminal protein (TP) and 5'-dAMP. Like the reaction in the presence of phi 29 DNA, TP.dAMP complex formation is strongly dependent on activating Mn2+ ions and on the efficient formation of a TP/DNA polymerase heterodimer. The nature of the TP-dAMP linkage was shown to be identical (a O-5'-deoxyadenylyl-L-serine bond) to that found covalently linking TP to the DNA of bacteriophage phi 29, indicating that this DNA-independent reaction actually mimics that occurring as the initiation step of phi 29 DNA replication. Furthermore, as in normal TP-primed initiation on the phi 29 DNA template, this novel reaction showed the same specificity for TP Ser232 as the OH donor and the involvement of the YCDTD amino acid motif, highly conserved in alpha-like DNA polymerases. However, unlike the reaction in the presence of phi 29 DNA, the DNA-independent deoxynucleotidylation of TP by the phi 29 DNA polymerase did not show dATP specificity, being possible to obtain any of the four TP.dNMP complexes with a similar yield. This lack of specificity together with the poor efficiency of this reaction at low deoxynucleoside triphosphate (dNTP) concentration reflect a weak, but similar stability of the four dNTPs at the phi 29 DNA polymerase dNTP-binding site. Thus, the presence of a director DNA would mainly contribute to stabilizing a complementary nucleotide, giving base specificity to the protein-primed initiation reaction. According to all these data, the novel DNA polymerase reaction described in this paper could be considered as a "non-DNA-instructed" protein-primed deoxynucleotidylation.     

phi29 DNA polymerase-terminal protein interaction. Involvement of residues specifically conserved among protein-primed DNA polymerases

By multiple sequence alignments of DNA polymerases from the eukaryotic-type (family B) subgroup of protein-primed DNA polymerases we have identified five positively charged amino acids, specifically conserved, located N-terminally to the (S/T)Lx(2)h motif. Here, we have studied, by site-directed mutagenesis, the functional role of phi29 DNA polymerase residues Arg96, Lys110, Lys112, Arg113 and Lys114 in specific reactions dependent on a protein-priming event. Mutations introduced at residues Arg96, Arg113 and Lys114 and to a lower extent Lys110 and Lys112, showed a defective protein-primed initiation step. Analysis of the interaction with double-stranded DNA and terminal protein (TP) displayed by mutant derivatives R96A, K110A, K112A, R113A and K114A allows us to conclude that phi29 DNA polymerase residue Arg96 is an important DNA/TP-ligand residue, essential to form stable DNA polymerase/DNA(TP) complexes, while residues Lys110, Lys112 and Arg113 could be playing a role in establishing contacts with the TP-DNA template during the first step of DNA replication. The importance of residue Lys114 to make a functionally active DNA polymerase/TP complex is also discussed. These results, together with the high degree of conservation of those residues among protein-primed DNA polymerases, strongly suggest a functional role of those amino acids in establishing the appropriate interactions with DNA polymerase substrates, DNA and TP, to successfully accomplish the first steps of TP-DNA replication.     

Functional domain for priming activity in the phage phi 29 terminal protein

By site-directed mutagenesis we have changed into Cys the Ser232 of the phi 29 terminal protein (TP) involved in the covalent linkage to dAMP for the initiation of replication. The mutant TP, highly purified, had about 0.7% of the priming activity of the wild-type (wt) protein p3. The linkage between the mutant protein p3 and dAMP was more labile to piperidine treatment than the serine-dAMP linkage in the wt protein p3, suggesting the presence of a different kind of linkage, Cys-dAMP. In the other three mutant TPs, residues Leu220, Ser223 and Ser226 were independently changed into Pro; the purified TP mutants had about 3%, 140% and 1% of the priming activity of the wt p3, respectively. All the mutant TP were able to interact with the phi 29 DNA polymerase and with DNA, suggesting that Leu220 and Ser226, in addition to Ser232, form part of a functional domain involved in the process of initiation of DNA replication.     

Two positively charged residues of phi29 DNA polymerase, conserved in protein-primed DNA polymerases, are involved in stabilisation of the incoming nucleotide

In DNA polymerases from families A and B in the closed conformation, several positively charged residues, located in pre-motif B and motif B, have been shown to interact with the phosphate groups of the incoming nucleotide at the polymerisation active site: the invariant Lys of motif B and the nearly invariant Lys of pre-motif B (family B) correspond to a His in family A DNA polymerases. In phi29 DNA polymerase, belonging to the family B DNA polymerases able to start replication by protein-priming, the corresponding residues, Lys383 and Lys371, have been shown to be dNTP-ligands. Since in several DNA polymerases a third residue has been involved in dNTP binding, we have addressed here the question if in the DNA polymerases of the protein-primed subfamily, and especially in phi29 DNA polymerase, there are more than these two residues involved in nucleotide binding. By site-directed mutagenesis in phi29 DNA polymerase the functional role of the remaining two conserved positively charged amino acid residues of pre-motif B and motif B (besides Lys371 and Lys383) has been studied. The results indicate that residue Lys379 of motif B is also involved in dNTP binding, possibly through interaction with the triphosphate moiety of the incoming nucleotide, since the affinity for nucleotides of mutant DNA polymerase K379T was reduced in DNA and TP-primed reactions. On the other hand, we propose that, when the terminal protein (TP) is present at the polymerisation active site, residue Lys366 of pre-motif B is involved in stabilising the incoming nucleotide in an appropriate position for efficient TP-deoxynucleotidylation. Although mutant DNA polymerase K366T showed a wild-type like phenotype in DNA-primed polymerisation in the presence of DNA as template, in TP-primed reactions as initiation and transition it was impaired, especially in the presence of the phi29 DBP, protein p6.     

Phi 29 DNA polymerase active site. Mutants in conserved residues Tyr254 and Tyr390 are affected in dNTP binding.

Phi 29 DNA polymerase shares with other alpha-like DNA polymerases several regions of amino acid similarity. Among them, the two conserved regions characterized by the amino acid motifs "D-NSLYP" and "K--NS(L/V)YG," regions 1 and 2a, respectively, according to Blanco et al. (Blanco, L., Bernad, A., Blasco, M. A. and Salas, M. (1991) Gene (Amst.) 100, 27-38) have been proposed to be part of the polymerization active site of alpha-like DNA polymerases. One phi 29 DNA polymerase mutant in residue Tyr254, located in conserved region 1, and two mutants in residue Tyr390, located in conserved region 2a, have been characterized. The three phi 29 DNA polymerase mutant proteins were affected in polymerization when Mg(2+)-dNTPs were used as substrate. However, when the substrate was Mn(2+)-dNTP, mutants behaved as the wild-type phi 29 DNA polymerase. Mutant Tyr254 to Phe (Y254F) was strongly affected in the protein-primed initiation step of phi 29 DNA replication showing a decreased affinity for Me(2+)-dATP, the initiating nucleotide. Furthermore, the analysis of the template-independent deoxynucleotidylation of the TP by Y254F mutant polymerase is consistent with a change in the relative affinity for dNTPs. On the other hand, mutants Y390F and Y390S were found to be hypersensitive to the dNTP analogs 2-(p-n-butylanilino)dATP and N2-(p-n-butyl-phenyl)dGTP. The results obtained indicate that residues Tyr254 and Tyr390 are involved, directly or indirectly, in Me(2+)-dNTP binding.     

Characterization and mapping of the pyrophosphorolytic activity of the phage phi 29 DNA polymerase. Involvement of amino acid motifs highly conserved in alpha-like DNA polymerases.

The phi 29 DNA polymerase, an alpha-like DNA polymerase, shows an inorganic pyrophosphate-dependent degradative activity with similar requirements to the corresponding one of Escherichia coli DNA polymerase I: (a) it requires a high concentration of inorganic pyrophosphate and is reversed by polymerization; (b) like DNA polymerization, it needs a duplex DNA with protruding 5' single-strand; (c) it acts in the 3' to 5' direction releasing free dNTPs, thus, it can be considered as the reversal of polymerization; (d) although a correctly base-paired 3' primer terminus is the preferred substrate, the pyrophosphorolytic activity is able to remove mismatched 3' ends. In agreement with the structural and functional model previously proposed for the phi 29 DNA polymerase, the analysis of point mutations has revealed that the pyrophosphorolytic activity, like the polymerization activity, is located at the C-terminal portion of the molecule, involving the amino acid motif YCDTD, highly conserved in alpha-like DNA polymerases. Furthermore, the analysis of phi 29 DNA polymerase mutants indicates that pyrophosphorolysis, like DNA polymerization, also requires an efficient translocation of the enzyme along the template.     

Regions at the carboxyl end of bacteriophage phi 29 protein p6 required for DNA binding and activity in phi 29 DNA replication.

Series of deletions corresponding to the carboxyl end of the phage phi 29 protein p6 have been constructed and their activity in the initiation of phi 29 DNA replication and their capacity to interact with the phi 29 DNA ends have been studied. Determination of the activity of the deletion mutants in phi 29 DNA replication indicated the dispensability of the 14 carboxy-terminal amino acids of the protein. The activity of protein p6 decreased with deletions from 23 to 39 amino acids and was undetectable when 44 amino acids were removed. A similar behaviour was obtained when the interaction of the mutant proteins with the phi 29 DNA ends was analyzed. These results indicate that the stimulation of phi 29 DNA replication by protein p6 requires a specific binding to the phi 29 DNA ends.     

Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication

The results presented in this paper indicate that the phi 29 DNA polymerase is the only enzyme required for efficient synthesis of full length phi 29 DNA with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. Analysis of phi 29 DNA polymerase activity in various in vitro DNA replication systems indicates that two main reasons are responsible for the efficiency of this minimal system: 1) the phi 29 DNA polymerase is highly processive in the absence of any accessory protein; 2) the polymerase itself is able to produce strand displacement coupled to the polymerization process. Using primed M13 DNA as template, the phi 29 DNA polymerase is able to synthesize DNA chains greater than 70 kilobase pairs. Furthermore, conditions that increase the stability of secondary structure in the template do not affect the processivity and strand displacement ability of the enzyme. Thus, the catalytic properties of the phi 29 DNA polymerase are appropriate for a phi 29 DNA replication mechanism involving two replication origins, strand displacement and continuous synthesis of both strands. The enzymology of phi 29 DNA replication would support a symmetrical model of DNA replication.     

Structural and functional studies on ø29 DNA polymerase

TheBacillus subtilis phage ø29 DNA polymerase, involved in protein-primed viral DNA replication, contains several amino acid consensus sequences common to other eukaryotic-type DNA polymerases. Using site-directed mutagenesis, we have studied the functional significance of a C-terminal conserved region, represented by the Lys-X-Tyr (“K-Y”) motif. Single point mutants have been constructed and the corresponding proteins have been overproduced and characterized. Measurements of the activity of the mutant proteins indicated that the invariant Lys and Tyr residues play a critical role in DNA polymerization. Interestingly, substitution of the invariant Lys either by Arg or Thr, produced enzymes with an increased or a largely reduced, respectively, capability to use a protein as primer, an intrinsic property of TP-priming DNA polymerases. On the other hand, the viral protein p6, which stimulates initiation of ø29 DNA replication by formation of a nucleoprotein complex at both DNA replication origins, increased (about 5-fold) the insertion fidelity of ø29 DNA polymerase during the formation of the TP-dAMP initiation complex. We propose a model in which the special strategy to maintain the integrity of the ø29 DNA ends, by means of a “sliding-back” mechanism, could also contribute to increase the fidelity of ø29 DNA replication.