JBC Origene Your Gene Company

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M303052200 on June 12, 2003

J. Biol. Chem., Vol. 278, Issue 35, 33482-33491, August 29, 2003
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
278/35/33482    most recent
M303052200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Truniger, V.
Right arrow Articles by Salas, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Truniger, V.
Right arrow Articles by Salas, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

{phi}29 DNA Polymerase Residue Leu384, Highly Conserved in Motif B of Eukaryotic Type DNA Replicases, Is Involved in Nucleotide Insertion Fidelity*

Verónica Truniger {ddagger}, José M. Lázaro, Miguel de Vega, Luis Blanco and Margarita Salas §

From the Instituto de Biologáa Molecular "Eladio Viñuela" (CSIC), Centro de Biologáa Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain

Received for publication, March 25, 2003 , and in revised form, June 2, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Replicative DNA polymerases achieve insertion fidelity by geometric selection of a complementary nucleotide followed by induced fit: movement of the fingers subdomain toward the active site to enclose the incoming and templating nucleotides generating a binding pocket for the nascent base pair. Several residues of motif B of DNA polymerases from families A and B, localized in the fingers subdomain, have been described to be involved in template/primer binding and dNTP selection. Here we complete the analysis of this motif, which has the consensus "KLX2NSXYG" in DNA polymerases from family B, characterized by mutational analysis of conserved leucine, Leu384 of {phi}29 DNA polymerase. Mutation of Leu384 into Arg resulted in a {phi}29 DNA polymerase with reduced nucleotide insertion fidelity during DNA-primed polymerization and protein-primed initiation reactions. However, the mutation did not alter the intrinsic affinity for the different dNTPs, as shown in the template-independent terminal protein-deoxynucleotidylation reaction. We conclude that Leu384 of {phi}29 DNA polymerase plays an important role in positioning the templating nucleotide at the polymerization active site and in controlling nucleotide insertion fidelity. This agrees with the localization of the corresponding residue in the closed ternary complexes of family A and family B DNA polymerases, contributing to form the binding pocket for the nascent base pair. As an additional effect, mutant polymerase L384R was strongly reduced in DNA binding, resulting in reduced processivity during polymerization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nucleotide selectivity by DNA polymerases primarily depends on the polymerization active site geometry, which is designed to accept Watson-Crick base pairs and reject base pairs differing from this geometry. dNTP binding induces conformational changes in the DNA polymerase that precede chemical changes and that are more than 10,000 times slower for incorrect than for correct dNTPs. These conformational changes position the incoming nucleotide for catalysis. Additionally, a slower rate of phosphodiester bond formation may further discriminate incorrect dNTPs (reviewed in Ref. 1). For the case in which misincorporation could not be prevented, most DNA polymerases contain, besides their DNA-primed 5'-3' polymerization activity, a 3'-5' exonuclease activity, which plays an important role in correcting incorporation errors, improving the fidelity of the replication process at least 2 orders of magnitude. {phi}29 DNA polymerase, a small 66-kDa monomeric enzyme belonging to the family of the eukaryotic-type DNA replicases (2, 3), also referred to as family B (4), has, in addition to these two activities, the ability to initiate {phi}29 DNA replication using a protein as primer (reviewed in Ref. 5). Whereas its DNA polymerization activity is highly accurate (discrimination factor between correct and incorrect nucleotides ranges from 104 to 106), protein-primed initiation of TP1-DNA has been shown to be rather inaccurate with a discrimination factor of only 102 (6). Initiation occurs opposite the second 3'-T residue of the TP-DNA template (7) and consists in the formation of a covalent phosphoester linkage between the hydroxyl group of Ser232 in the TP and 5'-dAMP (8) catalyzed by {phi}29 DNA polymerase in the presence of divalent metal ions (9). The product, TP-dAMP, is not degraded by the 3'-5' exonuclease activity (6). Therefore, the low fidelity of {phi}29 DNA polymerase during initiation would produce a high mutational rate on the initiation product. "Sliding back" of the initiation product in front of the first 3'-T residue of the template strand allows the second nucleotide to serve a second time as template for the following step, the TP-(dAMP)2 formation (7). This mechanism is proposed to control the fidelity of the initiation reaction. After some transition steps, {phi}29 DNA polymerase replicates each DNA strand, while displacing the other. Due to its high processivity (>70 kb) and strand displacement capacity, this DNA polymerase does not need the help of any helicase or processivity factor to replicate completely its 19-kb linear natural template, the TP-DNA (10, 11).

Three functional subdomains responsible for polymerization have been defined by comparison of the crystal structures of several DNA polymerases from families A and B following their structural similarity to a right hand: palm, fingers, and thumb (12). The palm contains the polymerization active site, while the thumb has been proposed to be important for DNA binding and processivity, and the fingers subdomain was shown to undergo substrate-induced conformational changes upon binding of both DNA and dNTP, rotating toward the palm to form a dNTP-binding pocket (reviewed in Refs. 13 and 14). In agreement with this, the highly conserved motif B (15), located in the fingers subdomain, was shown to be involved in both template/primer binding and dNTP selection in DNA polymerases from families A and B (see Fig. 1). The role of motif B (consensus sequence "KLX2NSXYG" in family B DNA polymerases) was first studied by site-directed mutagenesis in {phi}29 DNA polymerase: Tyr390 was shown to be involved in DNA-dependent dNTP binding (16) and proposed to be directly involved in checking base pairing correctness of the incoming nucleotide (17); mutations in Gly391 affected DNA binding, while mutations in Asn387 and Ser388 caused intermediate phenotypes (18); lately, the invariant Lys383 was shown to be one of the two residues that interact directly with the phosphate groups of the incoming nucleotide (19, 20).



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 1.
Location of motif B in the fingers subdomain. Polymerization domains of the crystal structures of RB69 and Klentaq DNA polymerases in the closed conformation. The subdomains are colored: fingers in green, thumb in blue, and palm in light pink. The substrates are colored: template strand in light purple, primer strand in light yellow, incoming nucleotide in dark yellow, while the templating nucleotide is colored in dark purple. The consensus sequence of motif B from family B DNA polymerases is "KLX2NSXYG" and that from family A DNA polymerases "KX4GX2YG." Some residues of this motif, located in the fingers subdomain, are colored and highlighted in the structural representation as follows: the invariant Lys is shown in orange, the invariant Tyr in yellow, and the second residue of the motif, Leu561 (RB69) and Thr664 (Klentaq), corresponding to Leu384 of {phi}29 DNA polymerase studied here, in red (crystallographic data from PDB1 IG9 (RB69 DNA polymerase) and PDB1 3KTQ [PDB] (Klentaq DNA polymerase).

 

In this study we complete the analysis of the KLX2NSXYG motif (Fig. 1) by assessing the functional importance of its second residue, Leu384 of {phi}29 DNA polymerase. The results indicate that this residue is important for positioning the templating nucleotide at the polymerization active site, thus controlling nucleotide insertion fidelity during template-directed TP-primed initiation and DNA-primed polymerization.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Nucleotides and Proteins—Unlabeled nucleotides were purchased from Amersham Biosciences. [{alpha}-32P]dATP (3000 Ci/mmol), and [{gamma}-32P]ATP (5000 Ci/mmol) were obtained from Amersham Biosciences. Restriction endo-nucleases were from New England Biolabs. T4 polynucleotide kinase was from Roche Applied Science.

{phi}29 TP-DNA was isolated according to Peñalva and Salas (21). The TP was purified as described by Zaballos and Salas (22). The {phi}29 single-stranded DNA-binding protein (SSB) and the double-stranded DNA-binding protein (DSB) p6, obtained from Bacillus subtilis cells infected with phage {phi}29, were purified as described previously (23, 24).

DNA Templates and Substrates—Oligonucleotides sp1 (5'-GATCACAGTGAGTAC), sp1c+18 (5'-ACTGGCCGTCGTTCTATTGTACTCACTGTGATC), sp1c+13 (5'-ACTGGCCGTCGTTGTACTCACTGTGATC), and sp1c+6 (5'-TCTATTGTACTCACTGTGATC) that have a 5'-extension of 18, 13, and 6 nucleotides, respectively, in addition to the sequence complementary to sp1, and the three variations of the last oligonucleotide (sp1c+6) differing in the first template base when hybridized to sp1 (position 6 from the 5'-end (underlined) is an A, C, or G residue), were obtained from ISOGEN. The oligonucleotide sp1 was first purified by electrophoresis on 8 M urea-20% polyacrylamide gels and then 5'-labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase. To analyze the exonuclease and polymerization activities on a template/primer structure, 5'-labeled sp1 was hybridized either to sp1c+18, sp1c+13 or sp1c+6, or any of its variations, in the presence of 0.2 M NaCl and 60 mM Tris-HCl, pH 7.5. To analyze processive DNA polymerization coupled to strand displacement by {phi}29 DNA polymerase, the universal primer (Roche Applied Science) was hybridized to M13mp8 ssDNA as described above, and the resulting molecule was used as a primer/template suitable for a rolling circle type of DNA replication. The sequences of oligonucleotides oriR29 (29-mer) and its complementary strand were identical to those of the right origin of {phi}29 DNA (7).

Site-directed Mutagenesis and Expression of {phi}29 DNA Polymerase Mutants—The wild-type {phi}29 DNA polymerase gene, cloned into M13mp19 (M13mp19w21; Ref. 25), was used for site-directed mutagenesis, carried out essentially as described previously (26), using the oligonucleotide-directed in vitro mutagenesis kit from Amersham Biosciences. The fragments carrying the different mutations were sub-cloned in plasmid pT7-4w2 (27), which expresses {phi}29 DNA polymerase under the control of the T7 RNA polymerase-specific {phi}10 promoter (28). The presence of the desired mutation and the absence of any other changes were confirmed by complete sequencing of each {phi}29 DNA polymerase mutant gene. Sequencing was carried out by the chain termination method, using Sequenase, version 2.0, from United States Biochemical Corp. and a set of synthetic oligonucleotides complementary to the {phi}29 DNA polymerase gene as sequencing primers. Expression of the mutant proteins was carried out in the Escherichia coli strain BL21(DE3) pLysS, which contains the T7 RNA polymerase gene under the control of the isopropyl-1-thio-{beta}-D-galactopyranoside-inducible lacUV5 promoter, and a plasmid constitutively expressing T7 lysozyme (29, 30). The purification of wild-type and mutant {phi}29 DNA polymerases was done as described by Lázaro et al. (27).

Buffers—The 10x reaction buffer (RB) used in all assays contained: 500 mM Tris-HCl, pH 7.5, 10 mM dithiothreitol, 40% glycerol, and 1 mg/ml bovine serum albumin. The buffer used for dilution of the polymerase contained: 25 mM Tris-HCl, pH 7.5, 120 mM NaCl, 1 mg/ml bovine serum albumin, and 50% glycerol.

DNA Gel Retardation Assay—The interaction of either wild-type or mutant {phi}29 DNA polymerase with a template/primer structure was assayed using the 5'-labeled sp1/sp1c+6 (15/21-mer) hybrid. The incubation mixture, in a final volume of 20 µl, contained 12 mM Tris-HCl, pH 7.5, 1 mM EDTA, 20 mM ammonium sulfate, 10 mM MgCl2, 0.1 mg/ml bovine serum albumin, 1.2 nM 5'-labeled sp1/sp1c+6, and, as indicated, a 1.2, 4.2, 8.4, or 16.8 nM concentration of either wild-type or mutant {phi}29 DNA polymerase (31). After incubation for 5 min at 4 °C, the samples were subjected to electrophoresis in 4% (w/v) polyacrylamide gels (80:1, monomer:bis), containing 12 mM Tris acetate, pH 7.5, and 1 mM EDTA, and run at 4 °C in the same buffer at 8 V/cm, essentially as described previously (32). After autoradiography, stable enzyme/DNA interaction was detected as a shift in the position of the free labeled DNA and quantitated by densitometry of the autoradiograms corresponding to different experiments.

Polymerase/3'-5' Exonuclease Coupled Assay—The hybrid molecule sp1/sp1c+6 (15/21 mer) contains a 6-nucleotide long 5'-protruding end, and therefore, the primer strand can be used both as substrate for the 3'-5' exonuclease activity and also for DNA-dependent DNA polymerization. The reaction mixture, in a final volume of 12.5 µl, contained: 1.25 µl of RB, 10 mM MgCl2, a 1.2 nM concentration of 5'-labeled hybrid molecule (sp1/sp1c+6), a 16.8 nM concentration of either wild-type or mutant {phi}29 DNA polymerase, and the indicated increasing concentrations of the four dNTPs. If indicated, after preincubation of the DNA polymerase with the labeled DNA (15/33-mer) for 10 min on ice, a 122-fold molar excess of the same but unlabeled hybrid molecule was added as trap at the same time as the reaction was started with Mg2+ and 1 µM dNTP. After incubation for 5 min at 25 °C, the reactions were stopped by addition of 3 µl of sequencing loading buffer. Samples were analyzed by 8 M urea-20% polyacrylamide gel electrophoresis and autoradiography. Polymerization or 3'-5' exonucleolysis were detected as an increase or decrease, respectively, in the size (15-mer) of the 5'-labeled sp1 primer. The exonucleolytic activity was calculated from the intensity of each degradation product, obtained by densitometry of the autoradiograms, corrected (multiplied) by the number of catalytic events giving rise to each of these degradation products. From these data, the catalytic efficiency (indicated in Table I) of each mutant derivative, assayed in linear conditions both in time and enzyme amount, was calculated relative to wild-type {phi}29 DNA polymerase.


View this table:
[in this window]
[in a new window]
  		TABLE I
Enzymatic activities of the mutant derivatives of {phi}29 DNA polymerase

The different activity assays were carried out with the indicated templates, as described under "Experimental Procedures." Numbers indicate percentage of activity obtained with the wild-type polymerase and is an average of several experiments. In the pol/exo coupled assay, the dNTP concentration required, in µM, to compete effectively the 3'-5' exonuclease activity is indicated. The factor of stimulation by p6 is given in parentheses. The slashes in the M13 replication separate polymerisation activity obtained in the absence (–) and presence (+) of SSB. The slashes in the TP-dAMP formation separate initiation activity obtained either with ds oriR29 or ss oriR29.

 

The analysis of the base specificity during DNA-primed polymerization was studied in two experiments: the first was a truncated polymerization experiment using the template/primer structure (sp1/sp1c+13) and 1, 5, 25, and 100 µM concentrations of dATP, dCTP, and dTTP. The reactions were performed as described above for the pol/exo-coupled assay, but incubated on ice to reduce exonucleolytic degradation. The second experiment was performed with the four possible variations of the template/primer structure (sp1/sp1c+6), differing only in the first template base (position 16), by separate addition of each of the four dNTPs at 100 µM and incubation on ice to reduce exonucleolytic degradation.

Replication of Primed M13-DNA—The reaction mixture, in a final volume of 25 µl, contained: 2.5 µl of RB, 10 mM MgCl2, a 20 µM concentration each of the four dNTPs, 13 nM [{alpha}-32P]dATP (2 µCi), 4.2 nM oligonucleotide-primed M13 ssDNA, and a 16.8 nM concentration of either wild-type or mutant {phi}29 DNA polymerase. If indicated, a 36 µM concentration of the {phi}29 SSB was added to the reaction. After incubation for the indicated times at 30 °C, the reaction was stopped by addition of 10 mM EDTA and 0.1% SDS. After filtration through Sephadex G-50 spin columns, the Cerenkov radiation of the excluded volume was determined to calculate the amount of incorporated dNMPs during this reaction. Elongation was studied by alkaline 0.7% agarose gel electrophoresis (33). The position of unit length {phi}29 DNA in the agarose gels was detected by ethidium bromide staining. The autoradiogram of the dried gels revealed the elongation efficiency.

Protein-primed Initiation Assay—The formation of TP-dAMP was performed either with or without TP-DNA as template. In both cases the reaction mixture, in a final volume of 25 µl, contained: 2.5 µl of RB, 20 mM ammonium sulfate, 0.1 µM [{alpha}-32P]dATP (2 µCi), 83.2 nM purified TP, and a 16.8 nM concentration of either wild-type or mutant {phi}29 DNA polymerase. When indicated, 34 µM p6 were added to the reaction. In the template-dependent assay (with 1.6 nM TP-DNA), 10 mM MgCl2 was used as metal activator, and the incubation time was for 5 min at 30 °C. The assay without template was performed with 1 mM MnCl2 as metal activator (known to reduce the Km for the initiating nucleotide of the {phi}29 wild-type DNA polymerase; Ref. 6), and the incubation was for 4 h at 30 °C. TP-primed initiation on an oligonucleotide (ss-oriR or ds-oriR, 29 mer) was performed as described by Méndez et al. (7) using the reaction mixture described above with a 72 nM concentration of the respective DNA polymerase, 154 nM TP, 0.85 µM oriR (ds or ss), 10 mM MgCl2 as the metal activator and incubating the samples for 10 min at 15 °C. Reactions were stopped by addition of 10 mM EDTA, 0.1% SDS, filtration through Sephadex G-50 spin columns (in the presence of 0.1% SDS), and further analysis by SDS-PAGE as described previously (21). The TP-dAMP complex was detected by autoradiography and quantitated by densitometric analysis. To calculate the Michaelis-Menten constant for dATP binding (Km and Vmax) in the TP-DNA initiation reaction, different dATP concentrations and incubation times were assayed. Initiation fidelity was studied in the presence of 1 mM MnCl2 and either 10 µM [{alpha}-32P]dATP, [{alpha}-32P]dCTP, [{alpha}-32P]dGTP, or [{alpha}-32P]dTTP (5 µCi). The assay was performed as described above.

TP-DNA Replication (Protein-primed Initiation, Transition, and Elongation)—The incubation mixture of the truncated replication experiments (in the absence of dCTP) contained, in a final volume of 25 µl: 2.5 µl of RB, 20 mM ammonium sulfate, 1.6 nM TP-DNA, a 83.2 nM concentration of purified TP, a 10 µM concentration of each dGTP, dTTP, and [{alpha}-32P]dATP (2 µCi), 10 mM MgCl2, a 16.8 nM concentration of the corresponding DNA polymerase, and 34 µM p6 when indicated. After incubation at 30 °C for 5 min the reactions were stopped by addition of 10 mM EDTA, 0.1% SDS, and the samples were filtered through Sephadex G50 spin columns in the presence of 0.1% SDS. Separation of TP-dAMP from TP-(dAMP)2 and the truncated transition products was carried out in SDS-containing 12% polyacrylamide gels (360 x 280 x 0.5 mm) as described previously (7) and detected by autoradiography. Quantitation was performed by densitometric scanning of the autoradiograms.

The incubation mixture of the TP-DNA replication reaction was as described above, but with a 20 µM concentration of the four dNTPs. The incubation time at 30 °C was 5, 15, and 60 min. The Cerenkov radiation in the excluded volume after filtration through Sephadex G50 spin columns was measured and used for quantitation. Elongation was studied by alkaline 0.7% agarose gel electrophoresis (33). The position of unit length {phi}29 DNA in the agarose gels was detected by ethidium bromide staining. The autoradiogram of the dried gels revealed the elongation efficiency.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Conservation of the Second Residue of the KLX2NSXYG Motif in DNA-dependent DNA Polymerases—The amino acid residue next to the invariant Lys of motif B, Leu384 in {phi}29 DNA polymerase, is highly conserved as a non-polar amino acid (Leu, Ile, Val, or Ala) in DNA-dependent DNA polymerases (19, 4). Only in two out of 35 family B DNA polymerases a non-conservative residue (Arg) can be found. Family A DNA polymerases show a preference for the non-polar Ala, Val, and Ile with four exceptions (three of them Thr) out of nine sequences. Because of its location in family A and family B DNA polymerases at the polymerization active site (Fig. 1) and its high conservation, especially in family B DNA polymerases, residue Leu384 of {phi}29 DNA polymerase (belonging to this group) was subjected to site-directed mutagenesis. Since this residue was conserved as non-polar amino acid in most of the other DNA polymerase sequences (Ile and Val), it was decided to substitute it with either a polar (Gln) or a positively charged (Arg) amino acid. These mutations were designed according to general suggestions for conservative substitutions (34) and secondary structure predictions (35, 36). The wild-type and mutant {phi}29 DNA polymerases were purified to homogeneity as described under "Experimental Procedures."

Leu384 of {phi}29 DNA Polymerase Is Involved in DNA Binding—The interaction of the wild-type and mutant {phi}29 DNA polymerases with a template/primer structure was studied by gel retardation assays (described under "Experimental Procedures"). In a native gel the wild-type enzyme bound to the template/primer structure results in a single retardation band (see Fig. 2), which most likely corresponds to a protein-DNA interaction in which the primer-terminus is stabilized at the polymerization active site (31). Both mutations in residue Leu384 caused a reduction in the DNA binding ability of {phi}29 DNA polymerase (see Fig. 2 and Table I). Whereas mutant L384Q was about 2-fold reduced in DNA binding capacity, mutant DNA polymerase L384R was especially affected in forming a stable DNA polymerase-DNA complex, resulting in retardation of the labeled DNA along the whole lane (Fig. 2). Similar results were obtained when a labeled ssDNA oligonucleotide was used (not shown). It should be noticed that the faint retardation band corresponding to the mutant DNA polymerase L384R-DNA complex run at a slightly different position than the wild-type DNA polymerase-DNA complex (Fig. 2). This fact reflects an abnormal binding that could be due to a change in the orientation/stabilization of the 3'-end of the substrate. An alteration like this could be induced by the loss of a specific contact with the hydrophobic portion of the nitrogen bases of the DNA or by the introduction of a longer and positively charged side chain in close proximity to the ssDNA binding region. The poor stability of the L384R-DNA complex, evidenced by the smeared retardation products, together with the altered mobility obtained could explain its reduced 3'-5' exonuclease activity (see Table I).



View larger version (65K):
[in this window]
[in a new window]
  		FIG. 2.
DNA binding capacity of mutant DNA polymerases analyzed by gel retardation. The 5'-labeled hybrid molecule sp1/sp1c+6 (15/21-mer) was incubated with increasing amounts of the wild-type or mutant {phi}29 DNA polymerases (see "Experimental Procedures"). After non-denaturating gel electrophoresis, the mobility of the free DNA (sp1/sp1c+6) and that of the polymerase-DNA complex was detected by autoradiography. Mean activity values relative to the wild-type are given in Table I.

 

Exonucleolysis to Polymerization Switching by {phi}29 DNA Polymerase Mutants at Leu384—The pol/exo balance not only depends on the polymerization and exonuclease activities, but also on the affinity of the polymerization active site for the nucleotides and on the partition of the primer strand between both active sites. Exonucleolysis to polymerization switching can be monitored by increasing the dNTP concentration progressively (polymerase/3'-5' exonuclease coupled assay, see "Experimental Procedures"). As it can be seen in Fig. 3, different dNTP concentrations are required for the wild-type {phi}29 DNA polymerase to start replication and polymerize the first nucleotide (25 nM, from position 15 to 16), to compete effectively the 3'-5' exonuclease activity replicating up to the penultimate nucleotide (100 nM, from position 16 to 20) and to replicate the last nucleotide of the template (1 µM, from position 20 to 21). Mutant DNA polymerase L384Q was able to perform the different polymerization steps at the same dNTP concentrations as the wild-type DNA polymerase (Fig. 3). Its exonuclease activity, determined as described under "Experimental Procedures," seemed to be slightly impaired (Table I) paralleling its mild defect in DNA binding. On the other hand, the exonuclease activity of mutant DNA polymerase L384R was strongly affected (Table I), paralleling its defect in DNA binding capacity. This mutant polymerase was able to polymerize the first nucleotide at a 25 times lower dNTP concentration (1 nM) than the wild-type DNA polymerase, but also needed 100 nM dNTPs to replicate the template/primer substrate from position 16 to 20 (Fig. 3). Interestingly, it was able to insert the 6th nucleotide in front of the last nucleotide of the template (position 21) at 100 nM dNTPs, a 10 times lower concentration than the wild-type DNA polymerase (see Fig. 3). A possible explanation for this polymerization phenotype of mutant DNA polymerase L384R could be that its reduced exonuclease activity favors especially the first and last polymerization steps. Additionally, this mutant polymerase could be affected in processive replication. To test this possibility, a more than 100 times molar excess of unlabeled DNA was added as competitor at the same time as the replication reaction was started with the metal ion, after preincubation of the DNA polymerase with the same but labeled template/primer molecule (15/33-mer, see "Experimental Procedures"). As can be seen in Fig. 4, both mutant DNA polymerases were affected in initial DNA binding and displayed a distributive elongation, as evidenced by the appearance of intermediate polymerization products (especially position 16).



View larger version (55K):
[in this window]
[in a new window]
  		FIG. 3.
DNA polymerase/exonuclease coupled assay. The assays were carried out as described under "Experimental Procedures," using the 32P-labeled hybrid molecule sp1/sp1c+6 (15/21-mer) as template/primer DNA. After autoradiography of the 8 M urea-20% polyacrylamide gel, polymerization or 3'-5' exonuclease are detected as an increase or decrease, respectively, in the size of the 5'-labeled sp1 primer. The positions of the non-elongated primer (15-mer), elongated primer (16-mer, 20-mer, and 21-mer), and degraded primer (4-mer) are shown. In lane c the DNA substrate was loaded alone as a control (15-mer). The dNTP concentration (nM) required to compete exonucleolysis, allowing efficient replication of the template, is shown in Table I for the wild-type and each {phi}29 DNA polymerase mutant.

 


View larger version (72K):
[in this window]
[in a new window]
  		FIG. 4.
Polymerization in the presence of a challenge DNA. Replication activity on the 15/33 mer template/primer structure with the wild-type and mutant polymerases in the absence (–) and presence (+) of a 122-fold excess of the same but unlabeled DNA as trap. This DNA trap was added after preincubation of the DNA polymerases with the labeled DNA substrate, at the same time as the reaction was started with the metal ion and 1 µM dNTP. The positions of the non-elongated primer (15-mer), elongated primer (16- and 33-mer), and degraded primer (4-mer) on the autoradiogram of the 8 M urea-20% polyacrylamide gel are shown.

 

Defective M13-DNA Replication Activity of the Mutant Polymerases Is Restored by {phi}29 SSB—Efficient polymerization on the long primed M13-ssDNA substrate used in this experiment requires the intrinsic processivity of {phi}29 DNA polymerase (10). As shown in Fig. 5, the wild-type DNA polymerase is able to replicate M13-DNA and to produce, because of its strand displacement capacity, DNA molecules greater than unit-length M13-DNA ("rolling circle" replication, see "Experimental Procedures"). Both mutant DNA polymerases were strongly affected in this assay (Fig. 5): mutant DNA polymerase L384R was impaired in reaching and passing the first round of replication (further replication would require strand displacement); mutant DNA polymerase L384Q, which showed wild-type-like activity on the shorter template/primer structure under multiple binding events (see Fig. 3), had a very low replication efficiency with this DNA, in parallel to the activity shown by the mutant on the shorter template/primer DNA under conditions of a single binding event (see Fig. 4). Premature products due to abortive stops could be detected with both mutant polymerases, possibly as the result of their reduced processivity during replication of this long ssDNA template with secondary structures (Fig. 5). In agreement with this, addition of {phi}29 SSB to this assay, in enough amount to cover all M13-ssDNA, restored to a great extent the defective replication activity of both mutant polymerases (Fig. 5, Table I).



View larger version (80K):
[in this window]
[in a new window]
  		FIG. 5.
Replication of primed M13-DNA. In the autoradiogram of the alkaline agarose gel shown, the position of unit-length M13-DNA (first round of replication) is indicated. The assays were carried out as described under "Experimental Procedures," using different incubation times at 30 °C, as indicated. When indicated, 36 µM {phi}29 SSB was added. Mean activity values relative to the wild-type are given in Table I.

 

Protein-primed Initiation Activity of the {phi}29 DNA Polymerase Mutants—{phi}29 DNA polymerase is able to covalently link any of the four dNMP residues to the TP in the absence of any template DNA (37). For this reaction the only interactions of the DNA polymerase required are those occurring with the TP and the initiating nucleotide (see "Experimental Procedures"). As shown in Fig. 6A, in the absence of any template the {phi}29 wild-type DNA polymerase catalyzes the formation of TP-dAMP. Mutant DNA polymerases L384R and L384Q showed nearly wild-type and 50% TP-deoxynucleotidylation activity, respectively (see also Table I), indicating that their capacity to interact with the TP and the initiating nucleotide was only slightly affected. In the presence of TP-DNA as template, where TP-deoxynucleotidylation is now templated by the second 3'-T residue, mutant DNA polymerase L384Q showed an initiation activity reduced to 55% of the wild-type DNA polymerase (Fig. 6A and Table I). Interestingly, a higher initiation activity than that of the wild-type DNA polymerase was observed with mutant DNA polymerase L384R (Fig. 6A and Table I). In agreement with these results, in the presence of TP-DNA the apparent affinity for the initiating nucleotide of mutant DNA polymerase L384R was 10 times higher than that of the wild-type enzyme (Table I). Under the same conditions the Vmax for mutant polymerases L384R and L384Q was 7- and 3-fold lower, respectively, than that of the wild-type enzyme (Table I). The lower Vmax of mutant DNA polymerase L384R explains why, despite its 10 times decreased Km, its initiation activity is only 1.4-fold higher than that of the wild-type enzyme. On the other hand, the Km and Vmax values for the initiating nucleotide in the absence of DNA template with mutant DNA polymerase L384R were wild-type like (Table I). Therefore, only in the presence of TP-DNA as template was the Km for the initiating nucleotide of mutant DNA polymerase L384R reduced. The initiation activity of mutant DNA polymerase L384R was also tested on different DNA templates: higher TP-dAMP formation than the wild-type DNA polymerase was found on 29-nucleotide-long DNA templates, single-stranded or double-stranded, containing the sequence of the right origin (oriR) of {phi}29 DNA (Table I).



View larger version (48K):
[in this window]
[in a new window]
  		FIG. 6.
TP-primed initiation, TP-(dAMP)2 formation and transition. A, template-independent (no template) and template-dependent (TP-DNA) formation of TP-dAMP (initiation) was comparatively studied using the wild-type and mutant DNA polymerases. The reactions were carried out as indicated under "Experimental Procedures." 34 µM DSB p6 was added when indicated (+). The TP-dAMP complex is seen as a band after autoradiography of the SDS-PAGE gel. Mean activity values relative to the wild-type are given in Table I. B, high resolution SDS-PAGE (as indicated under "Experimental Procedures") of truncated replication reactions carried out with Mg2+ as metal activator and a 10 µM concentration of either only dATP or dATP/dGTP/dTTP. 34 µM {phi}29 DSB p6 was added when indicated (+). The reaction with the wild-type DNA polymerase, and mutant polymerase L384R was incubated for 5 min at 30 °C, and with mutant polymerase L384Q for 10 min. The TP-dAMP (initiation), TP-(dAMP)2, and further truncated transition products appearing are indicated. The products theoretically expected due to the absence of dCTP during the first replication steps starting from the left (oriL) or right (oriR) end of {phi}29 TP-DNA (TP-(dNMP)8 and TP-(dNMP)11, respectively), and the one resulting from elongation of a misincorporation at position 12 from oriR (TP-dNMP14), are indicated on top of the sequence.

 

DSB protein p6, which activates the initiation of viral DNA replication, forms a nucleoprotein complex at both {phi}29 DNA ends, producing a conformational change in the DNA that probably leads to local opening of the DNA duplex (38) and favors the initiation reaction due to a decrease in the Km for the initiating nucleotide, dATP (39). As can be seen in Fig. 6A the initiation activity of the wild-type DNA polymerase increased 4.5 times upon addition of p6 (Table I). Surprisingly, addition of p6 caused a 30% reduction on the initiation activity of mutant DNA polymerase L384R and had only a stimulating effect of 1.7 times on the initiation activity of mutant DNA polymerase L384Q (Fig. 6A and Table I).

Protein-primed TP-DNA Replication Activity of the {phi}29 DNA Polymerase Mutants—TP-DNA replication not only requires the initiation reaction, but also several steps in which TP and DNA polymerase are still forming a complex. The transition from TP-primed initiation to DNA-primed replication requires elongation of the initiation product to about 10 nucleotides (40). To study the TP-(dAMP)2 formation, 10 µM dATP was used and the further transition steps were studied in a truncated replication assay, in the absence of dCTP (see "Experimental Procedures"). Fig. 6B shows that mutant DNA polymerase L384R was more efficient in TP-(dAMP)2 formation than the wild-type and mutant L384Q DNA polymerases. In agreement with this, the apparent affinity for dATP of mutant DNA polymerase L384R in TP-(dAMP)2 formation was also 10 times higher (Table I). As described by Blanco et al. (41), the wild-type {phi}29 DNA polymerase requires a higher dATP concentration for the TP-(dAMP)2 formation than for the initiation reaction, probably because the 3'-5' exonuclease activity can act on this product. Fig. 6B also shows that in the presence of dATP, dGTP and dTTP elongation by the wild-type DNA polymerase, due to the lack of dCTP, results in the formation of TP-(dAMP)8 and TP-(dAMP)11. A slight increase in the initiation, TP-(dAMP)2 formation and further transitions steps could be observed with the wild-type DNA polymerase upon addition of protein p6. Since protein p6 reduces the Km for the initiating nucleotide (39), and the experiments shown in Fig. 6B were performed at high dATP concentration (10 µM), the activation by p6 was not as high as that shown in Fig. 6A (0.1 µM dATP concentration). In the presence of p6 the initiation, TP-(dAMP)2 formation, and further transitions steps were slightly reduced with mutant DNA polymerase L384Q and were inhibited with mutant DNA polymerase L384R, especially the formation of the truncated transition products (Fig. 6B). Mutant DNA polymerase L384R seemed to incorporate an error at position 12 of oriR resulting in the decrease of TP-(dAMP)11 product and the appearance of TP-(dAMP)14.

The complete TP-DNA replication activity of the {phi}29 DNA polymerases was studied in the presence of all four nucleotides (see "Experimental Procedures"). The TP-DNA replication activities of the DNA polymerases mutated in residue Lys384 were low, 4 and 10% of the wild-type activity with mutant polymerase L384R and L384Q, respectively (Table I). The affected polymerization processivity could explain the reduced TP-DNA replication activity, despite the essentially normal initiation activity. In agreement with the results of the initiation and transition experiments, the replication of TP-DNA, especially of mutant DNA polymerase L384R, was reduced upon addition of p6 (see Table I). On the other hand, the presence of p6 did not affect the polymerization activity of mutant DNA polymerase L384R on other DNAs that do not bind p6 (template/primer structures and M13-DNA, not shown).

Nucleotide Selectivity of the {phi}29 DNA Polymerase Mutants in TP-primed Initiation and DNA-primed Polymerization Reactions—Nucleotide insertion fidelity during DNA-primed polymerization reactions was studied using different template/primer structures (see "Experimental Procedures"). Fig. 7A shows a limited elongation reaction (in the absence of dGTP) on a template/primer structure (15/28-mer); no misincorporation was observed with the wild-type and mutant L384Q DNA polymerases up to addition of a 100 µM concentration of dATP, dCTP, and dTTP. On the other hand, mutant DNA polymerase L384R inserted wrong nucleotides at position 19 at 5 µM dATP/dCTP/dTTP. The experiment shown in Fig. 7B was performed using the same template/primer structure as in the retardation and exo/pol experiments (15/21-mer) and adding each of the four dNTPs separately (at 100 µM, see "Experimental Procedures"). It can be observed that mutant polymerase L384R inserted errors independently of the nucleotide used, while mutant polymerase L384Q behaved as the wild-type enzyme. From experiments in which this template/primer structure differred in the first template base, it could be deduced that the misinsertion frequency of mutant DNA polymerase L384R was especially high when C or T residues were acting as template (not shown). Although these experiments were performed on ice, to keep the exonuclease activity as low as possible, the remaining exonuclease activity of the wild-type enzyme could be correcting the misinsertions it produces, while possibly the strongly affected exonuclease activity of mutant polymerase L384R would not. Therefore, it was important to study the nucleotide insertion fidelity during TP-primed initiation (see "Experimental Procedures"), since the 3'-5' exonuclease activity of {phi}29 DNA polymerase cannot act on the initiation product TP-dNMP and will not correct a misinsertion produced during initiation (6). Additionally, mutant DNA polymerase L384R had been shown to have an apparently higher affinity for the initiating nucleotide only in the presence of a DNA-template. Fig. 8 shows the dNTP usage relative to dATP in non-templated and templated initiation reactions. As can be seen for the wild-type DNA polymerase and both mutant DNA polymerases, dTTP was the preferred nucleotide for the non-templated TP-deoxynucleotidylation reaction. The relative usage of the four different dNTPs in this reaction was similar for all three DNA polymerases. Template-directed initiation occurs at both {phi}29 DNA ends in front of the second 3'-T residue of the TP-DNA template, so that dATP is the correct nucleotide for this reaction. As can be seen in Fig. 8 both mutant DNA polymerases (L384R more than L384Q) had higher error frequencies for the three incorrect nucleotides dCTP, dGTP, and dTTP than the wild-type DNA polymerase, indicating that in the presence of TP-DNA they had a lower discrimination capacity for incorrect nucleotides. Since the exonuclease activity cannot act on the initiation product, the lower base specificity of mutant DNA polymerases L384R and L384Q during initiation could not be the result of their affected exonuclease activity.



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 7.
Nucleotide insertion fidelity during DNA-primed polymerization. Fidelity experiments with wild-type and mutant DNA polymerases during DNA polymerization using two different template/primer structures (sequences indicated) were performed on ice, to reduce exonuclease activity, as described under "Experimental Procedures." A, the truncated replication reactions were performed at different dATP, dCTP, and dTTP concentrations, as indicated. The absence of dGTP results in a truncated product after correct replication of the first three nucleotides (position 18). Misincorporation results in incorporation of a further nucleotide (position 19). B, misincorporation activity was tested with each of the four dNTPs separately (at 100 µM), as indicated (dATP is the correct nucleotide). The positions of the non-elongated primer (15-mer) and elongated primer (16-mer, 17-mer) are shown.

 


View larger version (30K):
[in this window]
[in a new window]
  		FIG. 8.
Nucleotide insertion fidelity during TP-primed initiation. A, usage of dNTPs (dCTP, dGTP, and dTTP) relative to dATP (100%) of wild-type and mutant DNA polymerases during non-templated initiation (see "Experimental Procedures"). B, fidelity of the wild-type and mutant DNA polymerases during TP-DNA templated initiation (see "Experimental Procedures"). The dNTP usage is given as percentage of the correct TP-dAMP formed.

 

We can therefore conclude that during TP-primed TP-DNA initiation and DNA-primed polymerization reactions mutant DNA polymerase L384R and, to a lower extent, mutant polymerase L384Q have reduced nucleotide insertion fidelity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Comparison of the crystal structures of several DNA polymerases in their open and closed (with dsDNA and dNTP) conformations resulted in the observation that the fingers subdomain rotates toward the palm to form a nucleotide binding pocket (Klentaq (42), T7 DNA polymerase (43), rat DNA polymerase {beta} (44), and HIV-1 reverse transcriptase (45)). The only crystal structure of the replicating complex of a family B DNA polymerase that exists so far is the one of bacteriophage RB69 (46). By comparison of its open (47) and closed ternary conformations, a similar movement of the fingers subdomain toward the active site has been observed (46). The close proximity of palm and fingers, forming the pocket in which the nascent base pair can fit, results in a conformation that enhances base selection by DNA polymerases. Motif B, highly conserved in all DNA-dependent DNA polymerases, lies in the fingers subdomain (Fig. 1). In the crystal structure of RB69 DNA polymerase in its closed ternary conformation the residue corresponding to Leu384 of {phi}29 DNA polymerase studied here, Leu561, is at a distance of 3.6 Å from the templating nucleotide (46), while in the open conformation the distance is 7 Å (47), indicating that upon DNA and dNTP binding this residue comes nearer to the templating nucleotide at the polymerization active site (Fig. 9). Together with Tyr567 and Gly568 of the same motif it encloses the templating nucleotide (Fig. 9), and according to Franklin et al. (46) "it forms the only protrusion, which could interfere with a bulge on the major groove side of a mispair."



View larger version (27K):
[in this window]
[in a new window]
  		FIG. 9.
The Leu residue of motif B is located in the nascent base pair binding pocket. Crystal structure of the active site of the replicating complex of RB69 DNA polymerase (46). The incoming (yellow) and templating (gold) nucleotides are indicated. The following highly conserved residues of motif B are shown: Lys (blue), Leu (green), Asn (red), Ser (wine red), Tyr (mauve), and Gly (pink). The location of the Lys and Leu residues in the open conformation are indicated with lighter colors (crystallographic data from PDB1 IG9 (closed) and PDB1 H7 (open)).

 

The results obtained here with {phi}29 DNA polymerase lead us to propose that probably this protrusion provides discrimination against mispairs; mutation of the corresponding Leu384 resulted in mutant DNA polymerases with about two to three (L384Q) and eight (L384R) times increased mutation frequency during DNA-templated TP-primed initiation. This higher error frequency of the initiation reaction can be only due to reduced insertion fidelity, since the 3'-5' exonuclease cannot act on the initiation product (6). The relative usage of the four different dNTPs during template-independent initiation was similar for the wild-type and mutant DNA polymerases, indicating that the reduced nucleotide insertion fidelity was depending on the presence of a template and did not result from a directly increased dNTP binding capacity. The same conclusion could be drawn for the increased affinity for the initiating nucleotide of mutant DNA polymerase L384R, which was observed only in the presence of DNA as template (TP-DNA or oligonucleotides) but not in its absence. Possibly a different presentation of the templating nucleotide at the polymerization active site of mutant DNA polymerase L384R results in its apparently increased affinity for dATP, affecting also its reaction velocity during initiation. Protein p6, known to favor the initiation reaction due to a decrease in the Km for the initiating nucleotide (39) possibly due to a favorable presentation of the DNA template in front of the polymerization active site (48), may be incompatible with a different positioning of the templating nucleotide at the polymerization active site of mutant DNA polymerase L384R, resulting in its lower initiation and nearly no transition activity in the presence of p6. A role of residue Leu384 in presentation of the templating nucleotide at the polymerization active site is in agreement with the short distance between the Leu residue and the templating nucleotide in the crystal structure of RB69 DNA polymerase in its closed conformation (46). Mutation of this residue may affect the structure of the binding pocket for the nascent base pair at the side accommodating the templating nucleotide, causing its widening. Due to this, the positioning of the templating nucleotide and the control of correctness of the nascent base pair may be less strict, resulting in an apparently increased affinity for nucleotides due to a lower nucleotide insertion discrimination capacity and, as a consequence, reduced nucleotide insertion fidelity. This agrees with the increased error frequency observed with both mutant DNA polymerases during initiation with TP-DNA and with that observed with mutant DNA polymerase L384R during DNA-primed polymerization of template/primer structures. Widening of the binding pocket at the polymerization active site could result in less stable binding of the template strand, as observed especially with mutant DNA polymerase L384R, and explains the reduced processivity of the mutant DNA polymerases during polymerization. Competitor DNA was able to trap the mutant DNA polymerases bound to the labeled template/primer mainly before and after the first replication step. During replication of primed M13-DNA the impairment of the mutant DNA polymerases was relieved by the presence of {phi}29 SSB, indicating that the phenotype is due to delayed elongation and not the result of accumulation of misincorporations. Mutant DNA polymerase L384Q seemed to be especially impaired in initial binding of this long ssDNA template molecule containing secondary structures. The positive effect of {phi}29 SSB can be explained by its ssDNA-binding capacity: it covers all the M13-ssDNA and therefore concentrates binding of {phi}29 DNA polymerase to the dsDNA portion and inhibits the formation of secondary structures in this DNA template favoring elongation efficiency and strand displacement (49). It is important to stress that the change of only one residue of the polymerization active site of {phi}29 DNA polymerase, Leu384, results in reduction of the high intrinsic processivity of this enzyme.

Interestingly, comparison of the editing and replicating complexes of RB69 DNA polymerase indicates a movement of residues Lys560 and Leu561 (motif B). Therefore, mutation of the {phi}29 residue Leu384 could affect such a movement and, possibly as a consequence, the switching of the 3'-end of the primer strand from the polymerization to the exonuclease active site. This idea is in agreement with the reduction in the exonuclease activity of the mutant polymerases studied here.

The residues corresponding to Leu384 of {phi}29 DNA polymerase are especially highly conserved as non-polar amino acids (Leu, Ile, Val) in DNA-dependent DNA polymerases from family B (19). Therefore, and in agreement with its position in the crystal structure of RB69 DNA polymerase as a closed complex (3.6 Å from the templating nucleotide), the role of this residue in positioning the templating nucleotide and controlling nucleotide insertion fidelity could probably be extrapolated to all family B DNA polymerases. In DNA-dependent DNA polymerases belonging to family A the corresponding residue is less conserved. The crystallographic data from Taq DNA polymerase (family A) with template/primer DNA and ddCTP indicate that the corresponding residue Thr664 (see Fig. 1) interacts directly with the templating nucleotide (distance 3.1 Å; Ref. 42). Mutation of this polar residue into Arg or Pro also resulted in mutant DNA polymerases with low nucleotide insertion fidelity (50, 51), while mutation into Ser and Asn reduced replication fidelity only slightly, and mutation into Ile caused no change in the wild type mutation frequency (51). Therefore, non-polar (Leu) and polar (Thr) residues of different DNA polymerases from families A and B seem to be involved in the control of nucleotide insertion fidelity. Since the residues resulting from mutagenesis, Arg and even Gln, have longer side chains than Thr, Leu, Ile, Val, or Ala (and also than Ser and Asn), nucleotide insertion fidelity could be possibly controlled by steric complementarity between the protein and a correctly formed Watson-Crick base pair. This is in agreement with the finding that no H-bonds exist between the protein and the bases of the nascent base pair in the quaternary structure of T7 DNA polymerase (43) and the replicating complex of RB69 DNA polymerase (46). The change of Thr664 into Pro may have additionally a different effect, since Pro is a unique amino acid forming a ring structure. As has been shown for several DNA polymerases from families A and B, other residues forming the binding pocket for the nascent base pair are also involved in controlling nucleotide insertion fidelity as the Tyr of motif B (fingers subdomain; Refs. 16, 17, and 52–55) and the conserved Leu of motif A (palm subdomain; Refs. 56 and 57).

We conclude that the second amino acid residue of motif B of family A and B DNA polymerases, due to its position in the closed ternary complex in the binding pocket for the nascent base pair, plays an important role in controlling base-pairing correctness, possibly through optimal fit of the nascent base pair into this pocket. Mutation of this residue possibly affects the structure of the binding pocket for the nascent base pair at the side facing the templating nucleotide, resulting in different, less strict, positioning of the templating nucleotide at the polymerization active site and reduction of the control of nucleotide insertion fidelity, possibly achieved by steric interference.


   FOOTNOTES
 
* This work was supported by Research Grant 2R01 GM27242-23 from the National Institutes of Health, by Grant PB98-0645 from the Dirección General de Investigación Cientáfica y Técnica, and by an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular "Severo Ochoa." The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Postdoctoral fellow from the Comunidad Autónoma de Madrid. Present address: CEBAS, Campus Universidad de Murcia, 30100 Espinardo, Murcia, Spain. Back

§ To whom correspondence should be addressed. Tel.: 34-1-3978435; Fax: 34-1-3978490; E-mail: msalas{at}cbm.uam.es.

1 The abbreviations used are: TP, terminal protein; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; SSB, single-stranded DNA-binding protein; DSB, double-stranded DNA-binding protein. Back


   ACKNOWLEDGMENTS
 
We thank F. J. Esteban for his help in performing the site-directed mutagenesis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Kunkel, T. A., and Bebenek, K. (2000) Annu. Rev. Biochem. 69, 497–529[CrossRef][Medline] [Order article via Infotrieve]
  2. Bernad, A., Zaballos, A., Salas, M., and Blanco, L. (1987) EMBO J. 6, 4219–4225[Medline] [Order article via Infotrieve]
  3. Blanco, L., Bernad, A., Blasco, M. A., and Salas, M. (1991) Gene (Amst.) 100, 27–38[CrossRef][Medline] [Order article via Infotrieve]
  4. Braithwaite, D. K., and Ito, J. (1993) Nucleic Acids Res. 21, 787–802[Free Full Text]
  5. Salas, M. (1991) Annu. Rev. Biochem. 60, 39–71[CrossRef][Medline] [Order article via Infotrieve]
  6. Esteban, J. A., Salas, M., and Blanco, L. (1993) J. Biol. Chem. 268, 2719–2726[Abstract/Free Full Text]
  7. Méndez, J., Blanco, L., Esteban, J. A., Bernad, A., and Salas, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9579–9583[Abstract/Free Full Text]
  8. Hermoso, J. M., Méndez, E., Soriano, F., and Salas, M. (1985) Nucleic Acids Res. 13, 7715–7728[Abstract/Free Full Text]
  9. Blanco, L., and Salas, M. (1984) Proc. Natl. Acad. Sci. U. S. A. 82, 6404–6408
  10. Blanco, L., Bernad, A., Lázaro, J. M., Martín, G., Garmendia, C., and Salas, M. (1989) J. Biol. Chem. 264, 8935–8940[Abstract/Free Full Text]
  11. Blanco, L., and Salas, M. (1996) J. Biol. Chem. 271, 8509–8512[Free Full Text]
  12. Joyce, C. M., and Steitz, T. A. (1994) Annu. Rev. Biochem. 63, 777–822[CrossRef][Medline] [Order article via Infotrieve]
  13. Brautigam, C., and Steitz, T. A. (1998) Curr. Opin. Struct. Biol. 8, 54–63[CrossRef][Medline] [Order article via Infotrieve]
  14. Kunkel, T. A., and Wilson, S. (1998) Nat. Struct. Biol. 5, 95–99[CrossRef][Medline] [Order article via Infotrieve]
  15. Delarue, M., Poch, O., Tordo, N., Moras, D., and Argos, P. (1990) Protein Eng. 6, 461–467
  16. Blasco, M. A., Lázaro, J. M., Bernad, A., Blanco, L., and Salas, M. (1992) J. Biol. Chem. 267, 19427–19434[Abstract/Free Full Text]
  17. Saturno, J., Blanco, L., Salas, M., and Esteban, J. A. (1995) J. Biol. Chem. 270, 31235–31243[Abstract/Free Full Text]
  18. Blasco, M. A., Lázaro, J. M., Blanco, L., and Salas, M. (1993) J. Biol. Chem. 268, 16763–16770[Abstract/Free Full Text]
  19. Saturno, J., Lázaro, J. M., Esteban, F. J., Blanco, L., and Salas, M. (1997) J. Mol. Biol. 269, 313–325[CrossRef][Medline] [Order article via Infotrieve]
  20. Truniger, V., Lázaro, J. M., Esteban, F. J., Blanco, L., and Salas, M. (2002) Nucleic Acids Res. 30, 1482–1493
  21. Peñalva, M. A., and Salas, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 5522–5526[Abstract/Free Full Text]
  22. Zaballos, A., and Salas, M. (1989) Nucleic Acids Res. 17, 10353–10366[Abstract/Free Full Text]
  23. Martín, G., Lázaro, J. M., Méndez, E., and Salas, M. (1989) Nucleic Acids Res. 17, 3663–3672[Abstract/Free Full Text]
  24. Pastrana, R., Lázaro, J. M., Blanco, L., García, J. A., Méndez, E., and Salas, M. (1985) Nucleic Acids Res. 13, 3083–3100[Abstract/Free Full Text]
  25. Bernad, A., Blanco, L., Lázaro, J. M., Martín, G., and Salas, M. (1989) Cell 59, 219–228[CrossRef][Medline] [Order article via Infotrieve]
  26. Nakamaye, K., and Eckstein, F. (1986) Nucleic Acids Res. 14, 9679–9698[Abstract/Free Full Text]
  27. Lázaro, J. M., Blanco, L., and Salas, M. (1995) Methods Enzymol. 262, 42–49[Medline] [Order article via Infotrieve]
  28. Tabor, S., and Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1074–1078[Abstract/Free Full Text]
  29. Studier, F. W. (1991) J. Mol. Biol. 219, 37–44[CrossRef][Medline] [Order article via Infotrieve]
  30. Studier, F. W., and Moffatt, B. A. (1986) J. Mol. Biol. 189, 113–130[CrossRef][Medline] [Order article via Infotrieve]
  31. Méndez, J., Lázaro, J. M., Blanco, L., and Salas, M. (1994) J. Biol. Chem. 269, 30030–30038[Abstract/Free Full Text]
  32. Carthew, R. W., Chodosch, L. A., and Sharp, P. A. (1985) Cell 43, 439–448[CrossRef][Medline] [Order article via Infotrieve]
  33. McDonnell, M. W., Simon, M. N., and Studier, F. W. (1977) J. Mol. Biol. 110, 119–146[CrossRef][M