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

The linear genome of Bacillus subtilis phage f 29 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 f 29 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 f 29 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 1 , 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. The process of initiation of DNA implies, prior to nascent DNA synthesis, recognition of origins, unwinding of dsDNA, 1 and priming. These universal events require various DNA-protein and protein-protein interactions that widely dif-fer among replicons (reviewed in 1–3). One of the simplest models for origin and

The linear genome of Bacillus subtilis phage 29 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 29 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 29 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.
The process of initiation of DNA replication implies, prior to nascent DNA synthesis, recognition of origins, unwinding of dsDNA, 1 and priming. These universal events require various DNA-protein and protein-protein interactions that widely differ among replicons (reviewed in Refs. [1][2][3]. One of the simplest models for origin recognition and initiation of replication has been proposed for linear dsDNA genomes with a covalently attached TP. Free TP acts as a primer (primer TP) for DNA replication, remaining linked to the 5Ј ends of the fully replicated molecule (parental TP) to constitute the replication origins. TP-DNAs have been found in bacteriophages (e.g. 29, Nf, GA-1, Cp-1, and PRD1), animal viruses (e.g. adenoviruses), plasmids (e.g. S1, Kalilo), and bacteria (e.g. Streptomyces) (reviewed in Refs. 4

and 5).
Bacillus subtilis phage 29 initiates replication of its 19,285bp-long linear DNA by a protein-priming mechanism that has been extensively studied. Phage-encoded DNA polymerase and primer TP form a complex (pol⅐TP) that recognizes the ends of TP-DNA. Then DNA polymerase catalyzes the covalent linkage of dAMP to the OH group of Ser 232 of the TP, giving rise to the TP-dAMP initiation complex (reviewed in Ref. 4). The incorporation of the dAMP is directed by the Thy at the second position of the template strand, and full-length sequence is obtained by a sliding-back mechanism that aligns the Ade with the 3Јterminal Thy of the template (6). A similar mechanism has been described for the 29-related phage GA-1 (7), the Escherichia coli phage PRD1 (8), and adenovirus (9). 29 DNA polymerase elongates the initiation complex by a strand displacement mechanism in a very processive way, with no further requirements for any helicase or processivity factors (reviewed in Ref. 10). In addition to DNA polymerase and TP, phageencoded ss-and dsDNA-binding proteins (p5 and p6, respectively) are required for 29 DNA replication in vivo. Protein p5 stimulates replication in vitro (11) by interacting with the displaced single-stranded DNA (12). Protein p6, which has been proposed to play a role organizing and compacting the viral genome (13), activates the initiation of replication by forming multimeric nucleoprotein complexes at the ends of 29 DNA (14,15). No auxiliary host proteins have been described to be required for 29 DNA replication, unlike the case of adenoviruses, which also replicate by a protein-priming mechanism (reviewed in Refs. 16 and 17), where two cellular transcription factors, NFI and Oct-1, have been reported to interact with DNA polymerase and pTP (29 TP counterpart), respectively, directing the multiproteic complexes to the viral origins of replication (18 -20).
Synthesis of the 29 TP-dAMP complex in vitro requires the formation of a stable, equimolar, pol⅐TP complex (21) that recognizes the replication origins at the genome ends. The main signal to be recognized by the pol⅐TP complex for initiation of replication is the parental TP. Thus, when terminal DNA fragments lacking parental TP are used as templates, the initiation reaction becomes much less efficient, being about 10% of the activity obtained with TP-DNA (Ref. 22 and this work). Even in the absence of any template, DNA polymerase does retain the ability to deoxynucleotidylate TP unspecifically, although at a very low rate (23).
29 belongs to a family of phages classified into three groups (reviewed in Ref. 24) as follows: group A comprises phages BS32, 15, and PZA together with 29; group B includes M2, Nf, and B103, and group C has GA-1 as its sole member. The DNA polymerases of 29 and Nf are 572 amino acid residues long and share an 81.8% sequence identity (Ref. 25 and this work). The TPs of 29 and Nf have 266 amino acids with a 62.4% sequence identity (26,27). 29 TP complements in vivo PZA but not M2 TP sus mutants (28). Furthermore, in vitro assays showed that the 29 pol⅐TP complex is able to initiate DNA replication using PZA or 15 TP-DNAs as template but not Nf TP-DNA (22), suggesting the need of a TP-DNA from the same phage group for replication. The use of Mn 2ϩ instead of Mg 2ϩ in the in vitro system, which increases the formation of the initiation complex (Ref. 29 and this work), allowed us to detect initiation activity with pol⅐TP proteins from 29 or Nf and TP-DNAs from Nf or 29, respectively. This result prompted us to study the specificity of the template recognition by the initiation proteins (pol⅐TP). For this, we purified DNA polymerase and TP from the phage Nf to assay initiation in 29/Nf heterologous replication systems.
In this paper, we present evidence that the initiation activity is higher when the DNA polymerase is from the same phage as the TP-DNA, suggesting that a specific interaction between DNA polymerase and parental TP is essential for efficient initiation of replication and determines the specificity of origin recognition. DNA Templates-29 and Nf TP-DNAs (30) and proteinase K-digested 29 and Nf DNAs (31) were obtained as described. 29 and Nf DNA left terminal fragments (oriL), 259 and 263 bp, respectively, were obtained by PCR amplification from proteinase K-digested DNAs. Oligonucleotides were designed to produce a DraI restriction site to regenerate the genome end. In the case of 29, the PCR amplification product was phosphorylated and cloned in a SmaI-digested and dephosphorylated pBlueKScript cloning vector, giving rise to pL259. E. coli DH5␣ cells were transformed and plated on LB medium containing 100 g/ml ampicillin. Selection of pL259-containing colonies was carried out by assessing ␤-galactosidase activity. Transformants were grown in selective medium at 37°C, and 29 oriL was obtained after restriction of purified pL259 with DraI and EcoRV. This digestion results in the 259-terminal bp from 29 plus 18 additional bp from the plasmid. DNA fragments were purified from agarose gels using the Qia Quick Gel Extraction Kit of Qiagen.

Nucleotides, Oligonucleotides, and Enzymes-Unlabeled
Cloning, Expression, and Purification of 29 and Nf DNA Polymerases-29 DNA polymerase was obtained from E. coli NF2690 cells harboring plasmid pJLw2 and purified as described (32). Nf DNA polymerase gene was cloned and overproduced in E. coli cells. For this, phage Nf was obtained from infected B. subtilis cells and purified in a cesium chloride density gradient. Phage DNA was isolated by proteinase K treatment. The region encoding Nf DNA polymerase was amplified by PCR and digested with PvuII and EcoRI prior to cloning in a SmaI-EcoRI digested pT7-7 expression vector, under the control of the T7 RNA polymerase-specific 10 promoter (33). E. coli BL21 (DE3) pLysS cells (34) were transformed, and the cloned gene was sequenced by the dideoxynucleotide chain termination method (35) using a Sequenase kit. Cells containing the DNA polymerase gene were grown at 34°C in LB medium, in the presence of 100 g/ml ampicillin and 34 g/ml chloramphenicol, up to an optical density of 0.5 at 600 nm. T7 RNA polymerase expression was induced by addition of 1 mM isopropyl-1-thio-␤-D-galactopyranoside; after 20 min, 100 g/ml rifampicin were added and cells harvested 90 min later. Cells were disrupted by grinding with alumina and suspended in buffer 6 (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 7 mM ␤-mercaptoethanol, 5% glycerol) containing 0.5 M NaCl; alumina and cell debris were removed by centrifugation. DNA was precipitated by adding to the supernatant polyethyleneimine up to 0.25%, after adjusting absorbance at 260 nm to 120 units/ml. After centrifugation at 12,000 ϫ g for 10 min, ammonium sulfate was added to the supernatant to 70% saturation and centrifuged at 17,000 ϫ g for 30 min. The Nf DNA polymerase-containing pellet was resuspended in buffer 6 and applied to a phosphocellulose column at an ionic strength equivalent to 0.2 M NaCl. DNA polymerase was eluted with 0.45 M NaCl in buffer 6 and concentrated in another phosphocellulose column. Finally, DNA polymerase was obtained from blue dextran and heparinagarose columns by elution with 0.6 and 1.0 M NaCl in buffer 6, respec-tively. The sample was concentrated using a Centricon-30, and its purity was estimated to be about 90% by SDS-PAGE followed by Coomassie Blue staining.
Purification of 29 and Nf TPs-29 TP was purified from B. subtilis harboring a TP-expressing plasmid (28). To purify Nf TP B. subtilis 110NA cells were grown at 37°C in LB medium up to an optical density of 0.5 at 600 nm and infected with phage Nf at a multiplicity of 10. Cells were harvested 50 min after infection, and cell-free extracts were obtained as described above. After ammonium sulfate precipitation, TP was purified by phosphocellulose and calf thymus DNA-cellulose chromatography eluting at 0.5 M and 1 M NaCl in buffer 6, respectively. The protein was further purified through a heparin-agarose column, eluting at 1 M NaCl in buffer 6. The sample was concentrated with a Centricon-30. The protein was over 95% homogeneous as estimated by SDS-PAGE and Coomassie Blue staining.
DNA Polymerase-TP Interaction-Interaction between DNA polymerase (25 g) and TP (12.5 g) from 29 and Nf was analyzed by glycerol gradient centrifugation. The proteins, in 50 mM Tris-HCl, pH 7.5, 20 mM ammonium sulfate, 7 mM ␤-mercaptoethanol, 1 mM EDTA, 2 mM ZnSO 4 , 100 mM NaCl, and 8.5% glycerol were loaded on a 4-ml 15-30% glycerol gradient together with 25 g of BSA as molecular weight marker and centrifuged at 350,000 ϫ g for 24 h at 4°C. After centrifugation, 0.16-ml fractions were collected, and aliquots were analyzed on SDS-PAGE. Quantification was performed by densitometric scans of Coomassie-stained gels in a Molecular Dynamics 300A densitometer.
Interaction between DNA polymerase and TP from 29 and Nf was also analyzed by a DNA competition assay in which the 3Ј filling-in of a DNA fragment is inversely proportional to the amount of DNA polymerase interacting with TP. A dsDNA fragment with a 3Ј-recessive end, substrate for DNA polymerase, was obtained by restriction of pL259 with SpeI and PstI. DNA polymerase and TP from 29 and Nf were preincubated for 30 min at 4°C. The DNA fragment was added to a reaction mixture containing, in a 25-l final volume, 50 mM Tris-HCl, pH 7.5, 20 mM ammonium sulfate, 5% glycerol, 5% polyethylene glycol 1000, 1 mM dithiothreitol, 0.1 mg/ml BSA, 32 mM NaCl, 1 mM MnCl 2 , 33 nM [␣-32 P]dATP (2.5 Ci), and 400 nM of each dCTP, dTTP, and dGTP. The reaction was carried out for 2 min at 30°C and stopped by addition of 10 mM EDTA and 0.1% SDS. Samples were analyzed by SDS-PAGE.
Assay for Protein-primed Initiation of DNA Replication-The reaction mixture, in 25 l, was as described above except that the only deoxynucleotide present was 250 nM [␣-32 P]dATP (2.5 Ci); 1 mM MnCl 2 and the indicated amounts of purified DNA polymerase, TP, and template DNA were used. The samples were incubated at 30°C for the indicated times, and the reaction was stopped by addition of EDTA up to 10 mM. Samples were heated at 60°C for 5 min and digested with 25 units of micrococcal nuclease for 30 min at 37°C in 50 mM Tris-HCl, pH 8.8, and 22 mM CaCl 2 . The reaction was stopped as described above. Samples were filtered through Sephadex G-50 spin columns in the presence of 0.1% SDS and analyzed by SDS-PAGE. Quantification of the initiation complex formed was done by PhosphorImager densitometric scans of a exposed Fuji BAS-IIIs imaging plate.

RESULTS
Cloning and Sequencing of Nf DNA Polymerase Gene-To study the specificity of the protein-primed initiation of replication, we have used the in vitro systems of 29 and the related phage Nf. Partially purified samples containing both DNA polymerase and TP from Nf were used in a previous work (36); however, the current goal required the availability of each of the proteins free of the other. Since it was difficult to obtain DNA polymerase free of contaminant TP from Nf-infected B. subtilis cells, we cloned the Nf DNA polymerase gene, after PCR amplification of the corresponding region. Since phage M2 is derived from phage Nf by a deletion that does not affect the replication genes (37), we took advantage of the known sequence of the M2 phage DNA polymerase gene (gene G) (38) for the design of oligonucleotides. Nf DNA polymerase predicted gene was sequenced, and the product was identical in size and amino acid sequence to that of M2 DNA polymerase (38), except for codon 107, where arginine was found instead of lysine. The predicted protein had a molecular mass of 66.4 kDa and 81.8% identity and 91.3% homology to its 29 counterpart.
DNA Polymerase and TP from Phages 29 and Nf Are Able to Form Heterologous Complexes-Protein-primed initiation of DNA replication requires the prior formation of a complex between DNA polymerase and TP to recognize the replication origins (21). Studies on the specificity of the initiation reaction required the use of pol⅐TP complexes in which both proteins were from the same phage, 29 or Nf (hereafter homocomplexes), or one from 29 and the other from Nf (hereafter heterocomplexes). The formation of pol⅐TP complexes was analyzed by glycerol gradient centrifugation. TP (31 kDa) from either 29 (Fig. 1A) or Nf (Fig. 1B) sedimented behind the 67-kDa molecular mass marker BSA that cosedimented with the 29 and Nf DNA polymerases; however, when complexed with DNA polymerase, it sedimented slightly ahead of BSA, both in the case of 29 (Fig. 1C) and Nf (Fig. 1D) homocomplexes. A similar shift was observed when 29 and Nf heterocomplexes were analyzed (Fig. 1, E and F), although in the case of Nf DNA polymerase/29 TP about 40% of TP sedimented as free TP, suggesting they have a lower affinity (Fig. 1F); here, free and TP-complexed DNA polymerases are not resolved. Therefore, DNA polymerase and TP from the two phages are able to interact with each other although with different affinities. Nevertheless, when glycerol gradients were performed at 180 mM NaCl with a 10-fold lower amount of protein, pol⅐TP homo-but not heterocomplexes were detected (results not shown). This result indicates that DNA polymerase has a higher affinity for TP in homocomplexes than in heterocomplexes. The interaction between the different DNA polymerases and TPs was further tested in an assay in which DNA polymerase binding to TP is challenged by a dsDNA fragment with a recessive 3Ј end. Since TP binds to the same active site as a DNA primer in DNA polymerase (10) and they cannot bind simultaneously (39), the extent of the filling-in reaction would indicate the amount of free DNA polymerase, providing a qualitative measurement of the affinity of each DNA polymerase for each TP. The filling-in activities of both 29 (Fig. 2, lane b) and Nf (Fig. 2, lane f) DNA polymerases were basically the same; therefore, their activities are comparable in these conditions. The DNA primer-template competed more efficiently for 29 DNA polymerase when complexed with Nf TP (Fig. 2, lane e) than with 29 TP (Fig. 2, lane d). Likewise, Nf DNA polymerase was more efficiently competed when complexed with 29 TP (Fig. 2, lane i) than with Nf TP (Fig. 2, lane h). These results fully agree with the previous observation of the higher affinity of the DNA polymerases for the homologous TPs. Among the heterocomplexes, in agreement with the glycerol gradient centrifugation results, the affinity of the 29 polymerase for Nf TP (Fig. 2, lane e) is higher than that of the Nf polymerase for the 29 TP (Fig. 2, lane i). The assay was performed also with 10 mM Mg 2ϩ as catalytic metal, obtaining the same results; there- fore, the nature of the cation does not seem to affect the DNA polymerase-TP interaction.
The functionality of these complexes was tested by their ability to deoxynucleotidylate TP in the absence of template (23). Fig. 3 shows that, as expected, homocomplexes are more active than heterocomplexes, being the activity of the Nf homocomplex 64% that of the 29 counterpart. The activity of the heterocomplexes drops below 10% for the 29 pol/Nf TP and is negligible for Nf pol/29TP. The instability of the 29 TP/Nf DNA polymerase complex accounts for this low activity, as was shown by glycerol gradient centrifugation (Fig. 1F) and competition experiments (Fig. 2, lane i). Therefore, the differences observed in the activity of the heterocomplexes could, at least partially, reflect differences in the stability of the complexes.
Efficient Initiation of Replication Requires a Specific Recognition between DNA Polymerase and TP-DNA-The specificity of the protein-protein interactions involved in recognition of the origins of replication was studied by initiation of replication assays using 29 and Nf pol⅐TP homo-and heterocomplexes on both 29 and Nf TP-DNAs. When homocomplexes of one phage were assayed with TP-DNA of the other, the initiation activity was hardly detected when Mg 2ϩ was used as catalytic metal (results not shown). This problem was overcome by using Mn 2ϩ instead Mg 2ϩ , resulting in an increase of activity over 100-fold (not shown). The activity of either 29 or Nf DNA polymerase/TP homocomplexes on their respective TP-DNAs (Fig. 4, A  and B) is higher than that on TP-DNAs from the other phage. Thus, when 29 TP-DNA was assayed with the 29 pol⅐TP homocomplex, the activity detected after a 60-min reaction was 10-fold higher than that of Nf pol⅐TP homocomplex; moreover, the reaction with the latter was much slower, since this difference was about 200-fold after 5 min (Fig. 4A). On the other hand, when Nf TP-DNA was assayed with the Nf pol⅐TP homocomplex the activity was also higher than that with the 29 pol⅐TP homocomplex (Fig. 4B), but this difference was not as great as the one observed with 29 TP-DNA; this different behavior could be due to a lower specificity of 29 initiation proteins for the Nf TP-DNA or to a reduced stringency of the latter. Altogether, these results confirm the specificity between the pol⅐TP complex and the TP-DNA template.
To determine whether the recognition specificity of origins is provided by the DNA polymerase, the TP, or both, we assayed pol⅐TP heterocomplexes with 29 and Nf TP-DNAs as templates. Interestingly, we observed that when the DNA polymerase was from the same phage as the template, namely 29 pol/Nf TP with 29 TP-DNA (Fig. 4C) or Nf pol/29 TP with Nf TP-DNA (Fig. 4D), the activity was much higher than that found when the TP of the heterocomplex was from the same phage as the template, namely Nf pol/29 TP with 29 TP-DNA (Fig. 4C) or 29 pol/Nf TP with Nf TP-DNA (Fig. 4D).
These results strongly suggest that DNA polymerase provides the specificity of recognition of the replication origins, presumably by interaction with the parental TP. Initiation of TP-free Origins of Replication--The contribution of the parental TP in the initiation of protein-primed replication activity was assessed by comparison of 29 and Nf  (A and B), as well as 29 pol/Nf TP and Nf pol/29 TP heterocomplexes (C and D) were used. TP-dAMP formation is represented as arbitrary units derived from the absorbance of scanned bands in exposed gels and therefore comparable among the four panels. TP-DNAs with the corresponding TP-free left origins of replication (oriLs). Fig. 5A shows that the activity of 29 oriL is 12-20% of the activity of the TP-DNA preparation, as it has been described previously (22). The same situation was observed for Nf (Fig. 5B), although Nf showed an even higher dependence of parental TP, as Nf oriL activity is about 2-5% that of Nf TP-DNA. Thus, parental TP strongly enhances initiation of replication to a degree that depends on the system.
It had been described that 29 DNA polymerase and TP initiate replication with Nf oriL as template at about the same rate as with 29 oriL (22). Therefore, the specificity of recognition seemed to be lost with TP-free templates. The availability of purified DNA polymerase and TP from Nf, together with the use of Mn 2ϩ instead of Mg 2ϩ in the in vitro initiation reaction, allowed us to extend these studies measuring the rate of initiation of replication with pol⅐TP homo-and heterocomplexes with oriLs of 29 and Nf. With either 29 (Fig. 6A) or Nf (Fig. 6B) oriLs, the behavior of 29 and Nf homocomplexes was similar, and the specificity observed with TP-DNAs (see Fig. 4, A and B) is lost. With both templates the reaction with 29 pol⅐TP is faster than with Nf pol⅐TP, reaching a similar level of initiation at later times. When heterocomplexes were assayed, their activity was very low in all cases, due to the low intrinsic affinity of heterocomplexes (see Fig. 3) together with the low template activity of TP-free DNAs (see Fig. 5). We do not observe, as with TP-DNA (Fig. 4, C and D), the selective enhancement of initiation corresponding to the homology between DNA polymerase and template. These results confirm that the specific origin recognition by DNA polymerase involves mainly, if not exclusively, parental TP, whereas the nucleotide sequence by itself does not seem to play a significant role. DISCUSSION Linear DNA genomes with TPs, like that of B. subtilis phage 29, are replicated by a protein-priming mechanism. The origins of replication, located at the genome ends, are recognized by a complex formed by the DNA polymerase and a free TP that is the primer for the following replication round. Thus, the formation of the initiation complex involves the DNA polymerase and two TPs, one of them free, acting as a primer (primer TP) and the other covalently linked to the DNA 5Ј ends (parental TP). We have used both the proteins and the template DNA from 29 and the related phage Nf to study the specificity of the initiation reaction.
We first studied the interaction of DNA polymerases with TPs, since the formation of these complexes precedes the initiation of DNA replication. Heterologous DNA polymerases and TPs are able to form complexes, although their interaction, especially that of Nf DNA polymerase and 29 TP, is weaker than that of the homologous proteins. Since TP binds to the same active site in DNA polymerase as a primer DNA (10) and they cannot bind simultaneously (39), we challenged TP with DNA. The DNA was able to displace heterologous but not homologous TPs from DNA polymerases, indicating that homocomplexes are more stable than heterocomplexes, in agreement with the previous results. Accordingly, the ability of DNA polymerases to deoxyadenylylate a heterologous TP in the absence of any template was much lower than that with a homologous TP. However, their activities may not only reflect the stability of the different pol⅐TP complexes but also their catalytic efficiency.
29 DNA polymerase belongs to the eukaryotic polymerase ␣ family (40,41) of which the crystal structure of that of bacteriophage RB69, a member of the family, is known (42). The Ser 122 residue of 29 DNA polymerase, which has been proposed to be required for TP binding (39), is located in the exonuclease domain, facing the editing channel in the RB69 corresponding region (42). In addition, the motif Y 226 XG(G/A), which is also involved in TP binding, would be located near the polymerase-active site (43). On the other hand, the R 256 GD residues of 29 TP are involved in the interaction with DNA polymerase (44). The regions containing these interacting residues in both DNA polymerase and TP are highly conserved in Nf. Nevertheless, these phages may have evolved to get slight structural differences in their DNA polymerases and TPs that justify the preferential binding to their own partner.
Once the pol⅐TP complex is formed, it must specifically recognize the origins of replication. It has long been acknowledged as the critical role of the parental TP in the initiation reaction (30). The lack of activity of 29 pol⅐TP with TP-DNA templates from a different group of related phages, such as Nf (22) were assayed for initiation of replication with 50 ng each of DNA polymerase and TP from 29 or Nf, for the indicated times at 30°C. TP-dAMP formation is represented as arbitrary units derived from the densitometric scan of gel bands. than Mg 2ϩ (Ref. 29 and this work), allowed detection of initiation using 29 pol⅐TP complexes with Nf templates. Initiation activity with both 29 and Nf TP-DNAs was measured using 29 and Nf initiation proteins. As expected, pol⅐TP homocomplexes were more active with their own template. When pol⅐TP heterocomplexes were used, we found that the only type of template significantly active for initiation of replication was the TP-DNA from the same phage as the DNA polymerase. Since parental TPs readily interact with each other (45), it was assumed that primer TP would direct the pol⅐TP complex to the parental TP (44,46). Instead, our results point out that the specific recognition of parental TP by the DNA polymerase complexed with primer TP is essential for an efficient initiation of DNA replication. However, these results do not rule out a recognition between parental and primer TP, as has been recently proposed (47).
The selective recruitment of pol⅐TP complexes at the TPcontaining replication origins could provide the correct positioning of primer TP at the active site of polymerase. Nevertheless an optimized rate of initiation requires highly specific protein interactions and is only obtained when DNA polymerase is homologous to TP-DNA and primer TP. In fact, in vivo studies have shown a strict requirement for a primer TP homologous to TP-DNA and/or DNA polymerase (28,48).
Parental TP is essential for the efficient initiation of replication, as TP-free templates are much less active than TP-DNAs. Furthermore, TP-free templates show a similar activity with 29 and Nf pol⅐TPs, indicating that parental TP provides specificity to the template. In addition, these results further suggest that the nucleotide sequence by itself does not play a major role in the specific recognition of origins by the initiation proteins.
In conclusion, we can envisage a scenario for initiation of protein-priming replication in which the specificity of recognition of the replication origins is determined by the parental TP. The DNA polymerase engaged in a complex with primer TP specifically recognizes the parental TP. This recognition would result in a higher affinity binding of the pol⅐TP complexes to the origins and/or in an increase of their catalytic efficiency of the initiation reaction.