Phage phi 29 Terminal Protein Residues Asn80 and Tyr82 Are Recognition Elements of the Replication Origins*

Belén IllanaDagger , José M. Lázaro, Crisanto Gutiérrez, Wilfried J. J. Meijer, Luis Blanco, and Margarita Salas§

From the Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Initiation of phage phi 29 DNA replication starts with the recognition of the origin of replication, located at both ends of the linear DNA, by a heterodimer formed by the phi 29 terminal protein (TP) and the phi 29 DNA polymerase. The parental TP, covalently linked to the DNA ends, is one of the main components of the replication origin. Here we provide evidence that recognition of the origin is mediated through interactions between the TP of the TP/DNA polymerase heterodimer, called primer TP, and the parental TP. Based on amino acid sequence comparisons, various phi 29 TP mutants were generated at conserved amino acid residues from positions 61 to 87. In vitro phi 29 DNA amplification analysis revealed that residues Asn80 and Tyr82 are essential for functional interaction between primer and parental TP required for recognition of the origin of replication. Although these mutant TPs can form functional heterodimers with phi 29 DNA polymerase that are able to recognize the origin of replication, these heterodimers are not able to recognize an origin containing a mutant TP.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

DNA replication is a semiconservative process in which a DNA polymerase uses one DNA strand as a template for the synthesis of its complementary strand. All DNA polymerases require a preexisting primer to initiate DNA synthesis (1). In many cases, the primer is a short RNA or DNA molecule. In linear DNAs, which are unable to form circular or hairpin structures during replication, replication of the genome ends cannot take place via RNA or DNA priming. Several mechanisms have evolved to solve the problem of initiation of DNA replication in such linear genomes (2). In the case of viral linear genomes, the OH group of a serine, threonine, or tyrosine residue of a terminal protein (TP)1 molecule serves as a primer for DNA replication, and as a consequence, the TP molecule becomes covalently attached to the 5'-terminal nucleotide. This mechanism of initiation of replication is called protein priming (reviewed in Ref. 3). Once bound to the DNA, the TP may serve additional functions such as assisting in DNA packaging (4, 5), protection against nucleases (6), enhancement of infectivity (7, 8), stimulation of template activity (9), matrix attachment (10), stimulation of transcription (10), and recruitment of a new TP to the origin (11). As a step previous to the initiation of replication, TP interacts with the DNA polymerase to form a heterodimer (12). To distinguish between the different functions of TP, we will refer to the TP bound to the DNA ends as parental TP and the TP in the heterodimer with the DNA polymerase as primer TP. Interaction of primer TP and/or DNA polymerase with parental TP may increase the affinity of the TP/DNA polymerase heterodimer for the origin or could assist the latter for its correct positioning at the origin, which may be important for initiation of DNA replication at the correct position.

The protein-priming mechanism of DNA replication has been mainly studied in the Bacillus subtilis bacteriophage phi 29 and adenoviruses (see Refs. 2, 3, 13, and 14 for review). The phi 29 genome is a 19,285-base pair linear double-stranded DNA molecule (15) with a phage-encoded 31-kDa TP covalently attached to the 5' termini (16). Genetic studies and the development of an in vitro DNA replication system (17) led to the identification of the origins of replication at each end of the DNA molecule (18, 19). To activate the initiation of replication, dsDNA-binding protein p6 forms a nucleoprotein complex that would help to open the DNA ends (20). A primer TP/DNA polymerase heterodimer recognizes the origin of replication, probably through protein-protein interaction of the primer TP and/or phi 29 DNA polymerase with the parental TP. Then, the phi 29 DNA polymerase catalyzes the addition of the first dAMP (21) to the OH group provided by Ser232 of the primer TP (22). The formation of this first TP-dAMP is directed by the second nucleotide at the 3'-end of the template and then slides back one nucleotide to recover the terminal nucleotide (23). Following initiation, the same phi 29 DNA polymerase molecule completes replication of the parental strand.

phi 29 parental TP is an important requirement for in vitro initiation of phi 29 DNA replication (24). Two lines of evidence show that parental TP is also required in vivo. First, in a mixed infection experiment at 42 °C using phages that have a thermosensitive (ts) mutation in either gene 2 (encoding the DNA polymerase) or gene 3 (encoding TP), most of the phage progeny had the ts2 genotype (16), and second, successful transfection required an intact gene 3 product (17, 25). Moreover, formation of the TP-dAMP initiation complex was obtained using as template the TP-DNA isolated from the closely related Bacillus phage ø15 but not from the more distantly related Bacillus phage GA-1 or the pneumococcal phage Cp-1. The lack of activity in the initiation reaction of the TP-DNA isolated from these latter two phages could be because of differences in the parental TPs (24).

Structure-function studies and biochemical characterization of phi 29 TP provide a basis to gain insight about the different roles of this protein in phi 29 DNA replication. As shown here, a mutational analysis of phi 29 TP at conserved amino acids from positions 61 to 87 revealed that residues Asn80 and Tyr82 are important for recognition of the origin of replication. The results obtained indicate that parental TP plays an important role as a structural part of the replication origin through functional interactions with the primer TP.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

DNA, Proteins, and Chemicals-- [alpha -32P]dATP (3000 Ci/mmol) and unlabeled nucleotides were obtained from Amersham Pharmacia Biotech. TP-DNA was obtained as described (17). Proteinase K and DNA primers were from Roche Molecular Biochemicals. Reagents and materials for electron microscopy were from Balzers except benzyldimethylalkylammonium chloride, which was purchased from Bayer. Wild-type and mutant phi 29 TPs were purified as described by Zaballos et al. (26).

Amino Acid Sequence Comparison-- Multiple alignment of the TP sequences was performed in two steps. The selected protein sequences were aligned with the GAP program (27). Then, the alignments obtained were adjusted manually to find generally conserved regions or residues colinearly arranged.

Site-directed Mutagenesis and Expression of phi 29 TP Mutants-- The wild-type phi 29 TP gene, cloned into M13mp18, was amplified using the primer 5'-GAC ATC GAA TTC TAT TCA GAA GTT G-3' (sense) and 5'-CAT ATG CTG GAT CCT TTA ACG GAG C-3' (antisense). The site-directed mutagenesis was carried out by polymerase chain reaction using the primer 5'-GAC GCT TGT TCC ATC CAC TTA TTG-3' for mutant K61M, 5'-GAC GCT TGT TCC CTC CAC TTA TTG-3' for K61R, 5'-GAC GCT TGT TCC GTC CAC TTA TTG-3' for K61T, 5'-CAC ACC GTA TGC CCT CTT TTC GAA C-3' for N80S, 5'-CAC CAC ACC TAA TGC ATT CTT TTC G-3' for Y82L, 5'-CAC CAC ACC GGA TGC ATT CTT TTC G-3' for Y82S, 5'-CTA GCC ACC ACA TCG TAT GCA TTC-3' for G83D, and 5'-CTT AGC TTT ACT AGC CAC CAC ACC G-3' for S87R. The polymerase chain reaction fragments carrying the different mutations were subcloned in plasmid pAZe3s (28), which expresses phi 29 TP under the control of the lambda  PL promoter. The presence of the desired mutations and the absence of additional mutations were confirmed by sequencing each phi 29 TP mutant gene using the primers: 5'-GAC ATC ACG TTC TAT TC-3', 5'-GGA AGG AAC AAG CGT CC-3', 5'-GAC CCC ATG ATT TTG AC-3', and 5'-GAA TTT GAT AGT GAG GG-3'. Sequencing was carried out by the chain termination method (29), using Sequenase kit version 2.0 from U. S. Biochemical Corp. Expression of the mutant proteins was carried out in the Escherichia coli strain NF1.

phi 29 TP-dAMP Formation (Protein-primed Initiation Assay)-- The incubation mixtures contained (in 25 µl) 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, 20 mM ammonium sulfate, 4% glycerol, 0.1 mg/ml bovine serum albumin, 0.25 µM [alpha -32P] dATP (2.5 µCi), 0.5 µg of phi 29 TP-DNA, or 0.2 µg of single-stranded 29-mer oligonucleotide with the sequence corresponding to the right phi 29 DNA end (oriR(29), 5'-AAA GTA GGG TAC AGC GAC AAC ATA CAC CA-3'), 10 ng of wild-type or mutant phi 29 TP, and 20 ng phi 29 DNA polymerase. When indicated, oriR(29) was used as a template instead of TP-DNA, and 1 mM MnCl2 was used as a metal activator. After incubation for the indicated time at 30 °C (in conditions shown to be linear with time and enzyme amount), the reactions were stopped by adding EDTA to 10 mM and SDS to 0.1%. The samples were filtered through Sephadex G-50 spin columns in the presence of 0.1% SDS. The excluded volume was analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. The relative amounts of incorporated [alpha 32-P]dAMP were calculated by densitometric analysis of the autoradiographs.

phi 29 TP-DNA Replication Assay (Protein-primed Initiation Plus Elongation)-- The incubation mixtures contained (in 25 µl) 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 20 mM ammonium sulfate, 1 mM dithiothreitol, 4% glycerol, 0.1 mg/ml bovine serum albumin, 0.5 µg of TP-DNA, 20 µM each dCTP, dGTP, dTTP, and [alpha -32P]dATP (1 µCi), 10 ng of wild-type or mutant TP, and 20 ng of phi 29 DNA polymerase. After incubation for the indicated time at 30 °C, the reactions were stopped, and the samples were filtered as described in the previous paragraph. Quantitation of the DNA synthesized in vitro was carried out from the amount of radioactivity (Cerenkov radiation) corresponding to the excluded volume. The size of the synthesized DNA was determined by alkaline agarose gel electrophoresis followed by autoradiography.

phi 29 TP-DNA Amplification Assay-- The incubation mixtures contained (in 10 µl) 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 20 mM ammonium sulfate, 1 mM dithiothreitol, 4% glycerol, 0.1 mg/ml bovine serum albumin, 25 ng of TP-DNA, 80 µM each dCTP, dGTP, dTTP and [alpha -32P] dATP (1 µCi), 8 µg of protein p5 (ssDNA binding), 10 µg of protein p6, 20 ng of phi 29 DNA polymerase, and 10 ng of either wild-type or mutant TP. After incubation for 1.5 h at 30 °C, or at the indicated times, the reactions were stopped and the samples were filtered as described above. The DNA amplification factor was calculated as the ratio between the amount of DNA at the end of the reaction (input DNA plus synthesized DNA) and the amount of input phi 29 DNA. Quantitation of the DNA synthesized in vitro was carried out from the amount of radioactivity (Cerenkov radiation) measured from the excluded volume (2.66 nmol of dNTP incorporated, which correspond to 0.93 × 106 cpm, allows the synthesis of 887 ng of dsDNA). When indicated, the size of the amplified DNA was analyzed by either alkaline or native agarose gel electrophoresis followed by autoradiography and ethidium bromide staining.

Psoralen Cross-linking and Spreading of DNA Molecules for Electron Microscopy-- To analyze the structure of replicative intermediates produced during phi 29 DNA amplification in vitro, amplification reactions were stopped by adding 0.05 volume of 4,5',8-trimethylpsoralen (200 µg/ml in 100% ethanol), and the samples were maintained on ice for 5 min in the dark. Then, they were irradiated with 360 nm of ultraviolet light on ice for 30 min as described by Sogo and Thoma (30). After psoralen cross-linking, the samples were digested with proteinase K (500 µg/ml) for 2 h at 65 °C and extracted with phenol, and the DNA was precipitated with ethanol. Denaturation and spreading of the psoralen-cross-linked DNA for electron microscopy were carried out according to the benzyldimethylalkylammonium chloride technique as described by Sogo et al. (31). Electron micrographs were taken routinely at 80 kV at a magnification of × 10,000. Contour length measurements were carried out on photographic prints using a Summagraphic digitizer tablet.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Single Substitutions at Residues Lys61, Asn80, Tyr82, Gly83, and Ser87 in phi 29 TP Do Not Affect Its Function as a Primer-- A multiple alignment of the amino acid sequence of TPs of Bacillus sp. phages phi 29, PZA, Nf, B103 and GA-1, and TP of the E. coli phage PRD1 is presented in Fig. 1. These TPs are very similar in size, ranging from 258 (phage PRD1) to 267 (phage Nf) amino acids. The percentage of identical amino acids shared by these TPs is 9% (22% similarity) increasing to 32% (52% similarity) when only the TPs of Bacillus sp. are considered. Nonetheless, the TPs from the Bacillus sp. phages and from the E. coli phage PRD1 share a significant number of conserved amino acids, some of which may be important for its protein structure and/or function.


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Fig. 1.   Multiple alignment of the TP amino acid sequences of Bacillus sp. and E. coli bacteriophages. EMBL data base accession number are given in parentheses: phi 29 (J02479), PZA (M11813), Nf (Y00363), B103 (X99260), GA1 (X96987), and the E. coli bacteriophage PRD1 (M22161). Numbers at the beginning of the amino acid sequence refer to the position in the protein sequence. Black boxes enclose residues conserved in all sequences compared, and gray boxes enclose residues that are only conserved in the TPs of Bacillus bacteriophages. The following amino acids are considered conservative: S and T; A and G; K and R; D, E, Q, and N; I, L, M, V, Y, and F. The residues of phi 29 TP that have been subjected to mutagenesis are indicated with an asterisk.

To study the function of the most conserved residues in the TP region from position 61 to 87, single changes were introduced in two phi 29 TP residues that are invariant in all the aligned TPs shown in Fig. 1 (Lys61 and Ser87) and in three additional residues that are invariant in the TPs of the Bacillus phages (Asn80, Tyr82, and Gly83). The changes were designed taking into account secondary structure predictions (32, 33) and general suggestions for conservative substitutions (34). Eight mutants were obtained K61M, K61R, K61T, N80S, Y82L, Y82S, G83D, and S87R. Site-directed mutagenesis, overproduction, and purification of the mutant proteins were carried out as described under "Materials and Methods."

Replication of phi 29 TP-DNA starts at both ends by a specific protein-priming mechanism. In this process the viral DNA polymerase, which forms a heterodimer with the phi 29 TP, catalyzes the linkage of dAMP to the TP, which acts as a primer. To evaluate the primer function of mutant TPs, we studied the formation of the TP-dAMP initiation complex (initiation reaction) using as a template a single-stranded 29-mer oligonucleotide with the sequence corresponding to the right phi 29 DNA end (oriR(29)) but lacking parental TP (see "Materials and Methods"). As shown in Table I, the amount of dAMP incorporated to the various mutant TPs was comparable to that of the wild-type TP, indicating that these mutant TPs interact with phi 29 DNA polymerase and that they are able to serve as a primer in a reaction that does not involve interactions with parental TP.

                              
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Table I
Functions of wild-type and mutant ø29 TPs
Assays were carried out with 20 ng of ø29 DNA polymerase and 10 ng of ø29 wild-type (wt) or mutant TPs. Data represent the mean of the results obtained in three experiments.

To determine whether the presence of parental TP affects the primer function of the mutant TPs, the in vitro formation of the TP-dAMP initiation complex using phi 29 TP-DNA as a template was assayed with wild-type or the mutant TPs. The results were similar to those obtained when an oligonucleotide was used as a template (see Table I). Therefore, the interactions between the mutant TP/DNA polymerase heterodimer and the wild-type parental TP, which probably take place during the recognition of the origin in the initiation of replication, are not affected by these mutations.

After the formation of the TP-dAMP initiation complex, the DNA polymerase remains associated with the primer TP until a short DNA primer of 9 nucleotides has been formed. It is only after this so-called transition step that the two proteins dissociate, and the DNA polymerase continues elongation until complete replication of the nascent DNA chain is achieved (35). To study possible effects of the mutations introduced in the TP on the transition step, we carried out replication assays in which one full round of replication is allowed (see "Materials and Methods"). As shown in Fig. 2, the amount of replicated DNA and the velocity of the reactions were similar when either wild-type or mutant TPs was used (see also Table I). Therefore, the single amino acid changes introduced in the phi 29 TP do not affect the dissociation of the phi 29 TP/DNA polymerase heterodimer needed to proceed into elongation in the DNA replication process.


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Fig. 2.   In vitro phi 29 TP-DNA replication. The assays were carried out in the presence of 10 ng of either phi 29 wild-type (wt) or mutant TPs and 20 ng of phi 29 DNA polymerase (see "Materials and Methods"). After incubation for the indicated times at 30 °C, the samples were first used to calculate their relative replication activity by measuring the amount of incorporated [alpha -32P]dAMP (see Table I). Next, the samples were run in an alkaline agarose gel to determine the lengths of the synthesized DNAs. The migration position of unit length phi 29 DNA is indicated. bp, base pairs.

These results suggest that the mutant TPs are able to serve as a primer with an efficiency comparable to that of wild-type TP indicating that the residues Lys61, Asn80, Tyr82, Gly83, and Ser87 are not involved in the primer activity of the TP, and they are not involved in the transition step.

Mutations at the Asn80 and Tyr82 Residues Alter the Parental TP Function-- An in vitro phi 29 DNA amplification system has been described (36) that requires the following purified proteins of phi 29: TP, DNA polymerase, the dsDNA-binding protein p6, and the ssDNA-binding protein p5. During the first round of phi 29 TP-DNA amplification the TP present in the TP/DNA polymerase heterodimer acts as a primer and becomes covalently linked to the newly synthesized DNA strand. In subsequent replication rounds this TP molecule will act as a parental TP. This in vitro assay mimics the natural amplification of viral DNA. To analyze the possible effects of the mutations introduced in the TP on its function as parental TP, DNA amplification assays were carried out as described under "Materials and Methods." Fig. 3 shows the amount of DNA synthesized using wild-type or mutant TPs. Mutants K61M, K61R, K61T, G83D, and S87R led to levels of amplification similar or even higher than wild-type TP (see also Table I). However, mutants N80S, Y82L, and Y82S were very inefficient in the amplification reactions despite the fact that they have wild-type-like primer activity. To determine in which stage of the amplification process these mutant TPs are affected, the synthesis of DNA was studied as a function of time. DNA amplification experiments were carried out as described above with wild-type and mutant TPs N80S, Y82L, or Y82S. As shown in Fig. 4, the amount of DNA synthesized increased during the first 10 min, using as primer either the wild-type or mutant TPs. Afterward, DNA synthesis primed by the wild-type TP continued increasing with time, whereas DNA synthesis primed by mutant TPs begins to reach a plateau after 20 min. Analysis by native agarose gel electrophoresis showed that, in all cases, unit length phi 29 DNA was synthesized (Fig. 4B). The amount of de novo synthesized DNA (in nanograms) was quantified from the amount of radioactivity incorporated into DNA (Fig. 4A), and the amplification factor was calculated as the ratio between the amount of DNA at the end of the reaction (input DNA plus synthesized DNA) and the amount of input DNA. The data, shown in Table II, indicate that the amount of de novo synthesized DNA initiated with these mutant TPs implies an amplification factor close to 3 and never exceeded this value even after extended incubation times. Assuming that all DNA molecules were initiated and replicated completely, a 2-fold amplification factor is expected after the first round of replication in a normal process, becoming a 4-fold amplification factor after completion of the second round (see Fig. 5A). Control experiments with the wild-type TP showed amplification levels close to 30-fold (Table II). However, with mutant TPs N80S, Y82L, or Y82S the amplification factor only reached a maximum of 3, indicating that DNA synthesis was stalled in some way before completing a second round of replication. These results are explained in the scheme shown in Fig. 5B. After a wild-type TP-DNA molecule has been replicated in an in vitro experiment containing a mutant TP in the reaction mixture, both daughter molecules generated contain one origin of replication with a wild-type TP and the other with a mutant TP. If the presence of the mutant TP inactivates the corresponding origin, the second round of replication will be 50% productive because only the origin with a wild-type TP could be used for initiation. Therefore, if this model is correct, 3-fold is the maximal amplification factor expected in the assay. This is in agreement with the data obtained in the quantitation of the DNA synthesized in vitro (see Table II). Moreover, replication would stop after this incomplete second replication round because now all double-stranded origins would contain a mutant TP, being the DNA strands corresponding to the input DNA fully displaced. The possibility that the two displaced parental strands could hybridize to reconstitute a molecule with two active origins is precluded by the presence of phi 29 ssDNA- binding protein in the assay.


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Fig. 3.   In vitro phi 29 TP-DNA amplification with phi 29 wild-type or mutant TPs. The phi 29 TP-DNA amplification assays were carried out as described under "Materials and Methods," in the presence of 20 ng of phi 29 DNA polymerase and 10 ng of the indicated TP. The amount and size of amplified DNA was analyzed by alkaline agarose gel electrophoresis followed by ethidium bromide staining. The amount of input DNA is shown as control (C). wt, wild-type.


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Fig. 4.   Kinetics of in vitro phi 29 TP-DNA amplification with phi 29 wild-type or mutant TPs N80S, Y82L, or Y82S. A, DNA synthesized in vitro as a function of time. The amplification assay was carried out as described under "Materials and Methods," in the presence of 25 ng of phi 29 TP-DNA as template, 10 ng of phi 29 wild-type TP, or mutants N80S, Y82L, or Y82S. After incubation for the indicated times at 30 °C, the reaction was stopped and quantitated as the total amount (in nanograms) of dNTP incorporated, and (B) the sizes of the synthesized DNAs were analyzed by native agarose gel electrophoresis. The migration position of unit length phi 29 DNA is indicated. wt, wild type; bp, base pairs.

                              
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Table II
Kinetic analysis of in vitro ø29 DNA amplification
DNA amplification was assayed using 25 ng of ø29 TP-DNA as template in the presence of either 10 ng of ø29 wild-type (wt) TP or mutants N80S, Y82L, or Y82S, and 20 ng of ø29 DNA polymerase at the indicated times as described under "Materials and Methods." Numbers show the amplification factor calculated as the ratio between the amount of DNA at the end of the reaction (input DNA plus synthesized DNA) and the amount of input DNA. Data are the average of three experiments obtained with the Y82L TP or four experiments in the case of wt, N80S, and Y82S TPs.


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Fig. 5.   Model for in vitro phi 29 DNA amplification. Schematic phi 29 DNA amplification with wild-type TP (A) or mutant TPs N80S, Y82L, or Y82S (B). The different replicative intermediates produced during the first DNA replication round are depicted. Wild-type and mutant TPs are indicated in white and black, respectively. Bold lines represent the newly synthesized DNA strands, and plain lines represent parental DNA strands. RI, replicative intermediate.

Detection of Single-stranded DNA Molecules as a Consequence of the Inactivation of the Replication Origins-- The explanation depicted in Fig. 5B predicts the generation of full-size displaced ssDNA. To detect the presence of these ssDNA molecules, samples of the amplification assays (after 40 min) were subjected to electron microscopy analysis (see "Materials and Methods"). To distinguish between dsDNA and ssDNA molecules, a psoralen cross-linking procedure was used (30). It has been shown that psoralen treatment of phi 29 DNA in the presence of protein p6 followed by short UV light irradiation and DNA spreading under denaturing conditions produces molecules that contain small bubbles corresponding to ssDNA regions, in which the binding of p6 prevents psoralen cross-linking (37). Taking into account that protein p6 binds to dsDNA and not to ssDNA and that phi 29 ssDNA-binding protein covers the displaced strand of the replicative intermediate, molecules without bubbles correspond to displaced ssDNA. Fig. 6A shows a typical example of a replicative intermediate taken from a DNA amplification assay using the wild-type TP, and it consists of a dsDNA with one ssDNA branch that corresponds to the displaced strand from one origin. In addition to molecules with a single ssDNA branch, DNA molecules were observed with two ssDNA branches. However, no full-length phi 29 ssDNA molecules were observed in these samples (results not shown). Electron microscopy analysis of DNA amplification products using mutant TPs N80S, Y82L, or Y82S revealed the appearance of full-length phi 29 ssDNA molecules as shown in Fig. 6B for the mutant TP Y82L.


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Fig. 6.   Electron microscopy of in vitro cross-linked phi 29 DNA amplified either with wild-type TP (A) or with mutant Y82L TP (B). Samples were treated with psoralen and spread as described under "Materials and Methods." Denatured pUC18 DNA was used as a size marker. Short and long arrows point to dsDNA and ssDNA regions, respectively. Note that B shows a full-length phi 29 ssDNA molecule. Bars correspond to 500 base pairs.

Previous analysis of the different types of replicative intermediate molecules synthesized during phi 29 DNA replication indicated that neither in vivo (18) nor in vitro (38) full-length phi 29 ssDNA molecules were detected when natural phi 29 DNA template with TP at both DNA ends was used. Full-length ssDNA molecules were only observed in in vitro replication assays using recombinant phi 29 templates lacking TP at one end (38). Full-length phi 29 ssDNA molecules may also be generated when the initiation rate is reduced leading to only one initiation event/dsDNA molecule. However, as shown in Fig. 4, initiation rates of mutant TPs are similar to that of wild-type TP (at short reaction times). Therefore, the finding of full-length ssDNA molecules favors the model presented above in which an origin containing one of the mutant TPs N80S, Y82L, or Y82S is inactive.

Addition of Wild-type TP/DNA Polymerase Heterodimer Counteracts the Inactive Origin-- The above results show that mutations at residues N80 or Y82 of phi 29 TP do not affect the function of primer protein but alter function as parental TP. Thus, a mutant TP/DNA polymerase heterodimer is able to recognize a wild-type origin allowing a first round of DNA replication. However, once the mutant TP becomes parental TP the origin is inactive. This inactivation could be because of: (i) the involvement of these parental TP residues in a "one side" interaction with the primer TP/DNA polymerase heterodimer or (ii) a reciprocal ("two side") involvement of these residues of the parental-primer TP interactions. To distinguish between these two possibilities, we carried out the following experiments. After amplification for 40 min, when mutant primer TPs became mutant parental TPs and DNA synthesis stops, new TP/DNA polymerase heterodimers were added to the amplification reaction (Fig. 7). Upon the addition of wild-type TP/DNA polymerase heterodimer, renewed incorporation of dAMP was observed. On the contrary, addition of heterodimers with mutant TPs did not allow dAMP incorporation. These results indicate that the origin containing a mutant parental TP can be recognized (rescued) by a wild-type TP/DNA polymerase heterodimer but not when it contains a mutant TP. Therefore, the mutations introduced are critical when they are simultaneously present in both the primer and the parental TP, probably affecting their proper interaction required for a functional initiation reaction. To test this possibility, an amplification assay was carried out for 40 min using mutant TP N80S to obtain DNA molecules containing mutant TP at their origins. These products were then used as templates in initiation reactions with either wild-type or mutant TP/DNA polymerase heterodimers. Initiation reactions were only obtained with the wild-type heterodimer (results not shown). The capacity of the wild-type TP/DNA polymerase to rescue a mutant TP-DNA origin, both in amplification and initiation assays, seems to rule out the possibility that a mutant TP/DNA polymerase heterodimer forms an abortive (nonproductive) complex that blocks the mutant TP-DNA origin. Therefore, we consider that the inactivation of the origin is caused by the lack of functional interactions between mutant primer and mutant parental TP.


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Fig. 7.   In vitro phi 29 DNA amplification with mutant TPs N80S or Y82L is recovered by addition of wild-type TP/DNA polymerase heterodimer. First an amplification assay was carried out in the presence of 10 ng of mutant TPs N80S or Y82L for the indicated times at 30 °C (see "Materials and Methods"). After 40 min, TP/DNA polymerase heterodimers containing wild-type TP or mutant N80S or Y82L were added for a second amplification assay, in which the previously amplified DNA is used as template. The amount of incorporated [alpha -32P]dAMP was measured at the indicated times.

Conclusion-- Single substitutions at conserved residues Lys61, Asn80, Tyr82, Gly83, and Ser87 of phi 29 TP did not alter its primer function either in the presence or in the absence of parental TP in the template, indicating that these residues are not involved in (i) the interaction with phi 29 DNA polymerase and (ii) the formation of the linkage to dAMP. Furthermore, these mutant TPs were not affected in the transition step, a necessary step leading to TP/DNA polymerase dissociation to enable processive DNA polymerization. However, once the mutant primer TP became parental TP the replication origin was inactivated to initiate additional replication rounds.

Altogether, the results presented in this paper indicate that recognition of phi 29 DNA replication origins occurs through direct interactions between primer and parental TP. In addition, we have provided evidence that residues Asn80 and Tyr82 of phi 29 TP are essential for such functional interactions at the origins. Moreover, for the first time we have found mutations that do not affect the individual function of the TP either as primer or as parental but are critical when present simultaneously in the primer and parental TPs. These results support the existence of recognition elements at the phi 29 DNA replication origin that imply complementary interactions between primer and parental TP.

    ACKNOWLEDGEMENTS

We are very grateful to L. Villar for help during purification of proteins and to Dr. Maite Rejas for the electron microscopy work.

    FOOTNOTES

* This work was supported by research Grant 5RO1 GM27242-19 from the National Institutes of Health, by Grant PB93-0173 from the Dirección General de Investigación Científica y Técnica, by Grant ERBFMRX CT97 0125 from the European Economic Community, and by an institutional grant from Fundación Ramón Areces.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a predoctoral fellowship from Ministerio de Educación y Ciencia.

§ To whom correspondence should be addressed. Tel.: 3491-3978435; Fax: 3491-3978490; E-mail: msalas{at}cbm.uam.es.

    ABBREVIATIONS

The abbreviation used is: TP, terminal protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
  1. Kornberg, A., and Baker, A. (1992) DNA Replication, pp. 103-106, Freeman, San Francisco
  2. Salas, M., Miller, J. T., Leis, J., and DePamphilis, M. L. (1996) in Replication in Eukaryotic Cells (DePamphilis, M. L., ed), pp. 131-176, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  3. Salas, M. (1991) Annu. Rev. Biochem. 60, 39-71[CrossRef][Medline] [Order article via Infotrieve]
  4. Bjornsti, M. A., Reilly, B. E., and Anderson, D. L. (1982) J. Virol. 41, 508-517[Medline] [Order article via Infotrieve]
  5. Grimes, S., and Anderson, D. (1989) J. Mol. Biol. 209, 101-108[Medline] [Order article via Infotrieve]
  6. Dunsworth-Browne, M., Schell, R. E., and Berk, A. J. (1980) Nucleic Acids Res. 8, 543-554[Abstract]
  7. Hirokawa, H. (1972) Proc. Natl. Acad. Sci. U. S. A 69, 1555-1559[Abstract]
  8. Ronda, C., López, R., Gómez, A., and García, E. (1983) J. Virol. 48, 721-730[Medline] [Order article via Infotrieve]
  9. Pronk, R., Stuvier, M. H., and van der Vliet, P. C. (1992) Chromosoma (Berl.) 102, 39-45
  10. Schaack, J., Yew-Wai Ho, J., Freimuth, P., and Shenk, T. (1990) Gene (Amst.) 4, 1197-1208
  11. Gutiérrez, J., Vinós, J., Prieto, I., Méndez, E., Hermoso, J. M., and Salas, M. (1986) Virology 155, 474-483[Medline] [Order article via Infotrieve]
  12. Blanco, L., Prieto, I., Gutiérrez, J., Bernad, A., Lázaro, J. M., Hermoso, J. M., and Salas, M. (1987) J. Virol. 61, 3983-3991[Medline] [Order article via Infotrieve]
  13. Hay, R. T. (1996) in Replication in Eukaryotic Cells (DePamphilis, M. L., ed), pp. 699-719, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  14. van der Vliet, P. C. (1996) in Replication in Eukaryotic Cells (DePamphilis, M. L., ed), pp. 87-118, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  15. Vlcek, C., and Paces, V. (1986) Gene (Amst.) 46, 215-225[CrossRef][Medline] [Order article via Infotrieve]
  16. Salas, M., Mellado, R. P., Viñuela, E., and Sogo, J. M. (1978) J. Mol. Biol. 119, 269-291[Medline] [Order article via Infotrieve]
  17. Peñalva, M. A., and Salas, M. (1982) Proc. Natl. Acad. Sci. U. S. A 79, 5522-5526[Abstract]
  18. Inciarte, M. R., Salas, M., and Sogo, J. M. (1980) J. Virol. 34, 187-199[Medline] [Order article via Infotrieve]
  19. Sogo, J. M., García, J. A., Peñalva, M. A., and Salas, M. (1982) J. Virol. 116, 1-18
  20. Serrano, M., Salas, M., and Hermoso, J. M. (1990) Science 248, 1012-1016[Medline] [Order article via Infotrieve]
  21. Blanco, L., and Salas, M. (1984) Proc. Natl. Acad. Sci. U. S. A 81, 5325-5329[Abstract]
  22. Hermoso, J. M., Méndez, E., Soriano, F., and Salas, M. (1985) Nucleic Acids Res. 13, 7715-7728[Abstract]
  23. Méndez, J., Blanco, L., Esteban, J. A., Bernad, A., and Salas, M. (1992) Proc. Natl. Acad. Sci. U. S. A 89, 9579-9583[Abstract]
  24. García, J. A., Peñalva, M. A., Blanco, L., and Salas, M. (1984) Proc. Natl. Acad. Sci. U. S. A 81, 80-84[Abstract]
  25. Salas, M., García, J. A., Peñalva, M. A., Blanco, L., Prieto, I., Mellado, R. P., Lázaro, J. M., Pastrana, R., Escarmís, C., and Hermoso, J. M. (1983) in Mechanisms of DNA Replication and Recombination (Cozzarelli, N. R., ed), pp. 203-223, Alan R. Liss Inc., New York
  26. Zaballos, A., Lázaro, J. M., Méndez, E., Mellado, R. P., and Salas, M. (1989) Gene 83, 187-195[CrossRef][Medline] [Order article via Infotrieve]
  27. Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395[Abstract]
  28. Zaballos, A., Salas, M., and Mellado, R. P. (1987) Gene (Amst.) 58, 67-76[Medline] [Order article via Infotrieve]
  29. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A 74, 5463-5467[Abstract]
  30. Sogo, J. M., and Thoma, F. (1989) Methods Enzymol. 170, 142-165[Medline] [Order article via Infotrieve]
  31. Sogo, J. M., Stasiak, A., DeBernardin, W., Losa, R., and Koller, T. (1987) in Electron Microscopy in Molecular Biology: A Practical Approach (Sommerville, J., and Scheer, U., eds), pp. 61-79, IRL Press, Oxford
  32. Chou, P. I., and Fasman, G. D. (1978) Adv. Enzymol. Relat. Areas Mol. Biol. 47, 45-148[Medline] [Order article via Infotrieve]
  33. Garnier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120, 97-120[Medline] [Order article via Infotrieve]
  34. Bordo, D., and Argos, P. (1991) J. Mol. Biol. 217, 721-729[Medline] [Order article via Infotrieve]
  35. Méndez, J., Blanco, L., and Salas, M. (1997) EMBO J. 16, 2519-2527[Abstract/Free Full Text]
  36. Blanco, L., Lázaro, J. M., de Vega, M., Bonnin, A., and Salas, M. (1994) Proc. Natl. Acad. Sci. U. S. A 91, 12198-12202[Abstract/Free Full Text]
  37. Gutiérrez, C., Freire, R., Salas, M., and Hermoso, J. M. (1994) EMBO J. 13, 269-276[Abstract]
  38. Gutiérrez, C., Sogo, J. M., and Salas, M. (1991) J. Mol. Biol. 222, 983-994[Medline] [Order article via Infotrieve]


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