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

del Prado A, Villar L, de Vega M, Salas M - Nucleic Acids Res. (2011)

Bottom Line: The 3D structure of the DNA polymerase/TP heterodimer allowed the identification of TP residues that could be responsible for interaction with the DNA polymerase.Here, we examined the role of TP residues Arg158, Arg169, Glu191, Asp198, Tyr250, Glu252, Gln253 and Arg256 by in vitro analyses of mutant derivatives.The results showed that substitution of these residues had an effect on either the stability of the TP/DNA polymerase complex (R158A) or in the functional interaction of the TP at the polymerization active site (R169A, E191A, Y250A, E252A, Q253A and R256A), affecting the first steps of Φ29 TP-DNA replication.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular Eladio Viñuela (CSIC), Centro de Biología Molecular Severo Ochoa (CSIC-UAM), C/Nicolás Cabrera 1, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.

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

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(A) In vitro protein-primed initiation with TP mutants. The initiation assay was performed as described in ‘Materials and Methods’ section, in the presence of 10 ng of either wild-type or the indicated mutant TP, 20 ng of DNA polymerase and 500 ng of ϕ29 TP-DNA. After 4 min incubation at 30°C, the reactions were stopped, processed and analysed by SDS–PAGE and autoradiography. The position of the TP–dAMP complex is indicated. (B) Competition for DNA polymerase between wild-type and mutant TPs. The assay of formation of TP-dAMP in the absence of TP-DNA by the wild-type ϕ29 TP/DNA polymerase heterodimer was performed in the presence of increasing amounts of TP mutants (the reaction conditions are described under ‘Materials and Methods’ section). Reactions were started by adding 1 mM MnCl2 and, after incubation for 90 min at 30°C, were stopped and analysed as indicated for the protein-primed initiation assay. The TP-dAMP formed in the different competition conditions relative to that formed in the absence of competition (100%) is indicated. As control of competition, the inactive TP mutant S232C was used. Data are represented as mean ± SD corresponding to three independent experiments.
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gkr1283-F2: (A) In vitro protein-primed initiation with TP mutants. The initiation assay was performed as described in ‘Materials and Methods’ section, in the presence of 10 ng of either wild-type or the indicated mutant TP, 20 ng of DNA polymerase and 500 ng of ϕ29 TP-DNA. After 4 min incubation at 30°C, the reactions were stopped, processed and analysed by SDS–PAGE and autoradiography. The position of the TP–dAMP complex is indicated. (B) Competition for DNA polymerase between wild-type and mutant TPs. The assay of formation of TP-dAMP in the absence of TP-DNA by the wild-type ϕ29 TP/DNA polymerase heterodimer was performed in the presence of increasing amounts of TP mutants (the reaction conditions are described under ‘Materials and Methods’ section). Reactions were started by adding 1 mM MnCl2 and, after incubation for 90 min at 30°C, were stopped and analysed as indicated for the protein-primed initiation assay. The TP-dAMP formed in the different competition conditions relative to that formed in the absence of competition (100%) is indicated. As control of competition, the inactive TP mutant S232C was used. Data are represented as mean ± SD corresponding to three independent experiments.

Mentions: ϕ29 DNA replication starts from both origins placed at the termini of the linear ϕ29 TP-DNA molecule. The DNA polymerase forms a heterodimer with a free TP molecule and catalyses the template-directed insertion of 5′ dAMP onto the hydroxyl group of TP Ser232. To study the primer function of mutant TPs, the formation of the TP-dAMP complex (initiation reaction) was evaluated using as template ϕ29 TP-DNA. As shown in Figure 2A and Table 1, there were mutants severely (R158A) or moderately (R169A, Q253A and R256A) impaired in the formation of the TP-dAMP product. As the initiation of ϕ29 TP-DNA replication is a template-directed event, such deficiency could be a consequence of a faulty recognition/binding of the replication origins. To study this possibility, we made use of the ability exhibited by ϕ29 DNA polymerase to catalyse the deoxynucleotidylation of the TP in the absence of template, the formation of the TP-dAMP product relying exclusively on a functional DNA polymerase-TP interaction (24). As shown in Table 1, except for mutant D198A, the rest of the TP derivatives showed a drop in their priming ability. Thus, no activity was detected with mutants R158A, R169A, E252A, Q253A and R256A, and TP derivatives E191A and Y250A showed a decrease of 3- and 7-fold, respectively, in their priming activity relative to the template-directed reaction. Thus, the low TP-deoxynucleotidylation levels exhibited by TP mutants could be pointing to a defective interaction with the DNA polymerase.Figure 2.


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

del Prado A, Villar L, de Vega M, Salas M - Nucleic Acids Res. (2011)

(A) In vitro protein-primed initiation with TP mutants. The initiation assay was performed as described in ‘Materials and Methods’ section, in the presence of 10 ng of either wild-type or the indicated mutant TP, 20 ng of DNA polymerase and 500 ng of ϕ29 TP-DNA. After 4 min incubation at 30°C, the reactions were stopped, processed and analysed by SDS–PAGE and autoradiography. The position of the TP–dAMP complex is indicated. (B) Competition for DNA polymerase between wild-type and mutant TPs. The assay of formation of TP-dAMP in the absence of TP-DNA by the wild-type ϕ29 TP/DNA polymerase heterodimer was performed in the presence of increasing amounts of TP mutants (the reaction conditions are described under ‘Materials and Methods’ section). Reactions were started by adding 1 mM MnCl2 and, after incubation for 90 min at 30°C, were stopped and analysed as indicated for the protein-primed initiation assay. The TP-dAMP formed in the different competition conditions relative to that formed in the absence of competition (100%) is indicated. As control of competition, the inactive TP mutant S232C was used. Data are represented as mean ± SD corresponding to three independent experiments.
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Related In: Results  -  Collection

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gkr1283-F2: (A) In vitro protein-primed initiation with TP mutants. The initiation assay was performed as described in ‘Materials and Methods’ section, in the presence of 10 ng of either wild-type or the indicated mutant TP, 20 ng of DNA polymerase and 500 ng of ϕ29 TP-DNA. After 4 min incubation at 30°C, the reactions were stopped, processed and analysed by SDS–PAGE and autoradiography. The position of the TP–dAMP complex is indicated. (B) Competition for DNA polymerase between wild-type and mutant TPs. The assay of formation of TP-dAMP in the absence of TP-DNA by the wild-type ϕ29 TP/DNA polymerase heterodimer was performed in the presence of increasing amounts of TP mutants (the reaction conditions are described under ‘Materials and Methods’ section). Reactions were started by adding 1 mM MnCl2 and, after incubation for 90 min at 30°C, were stopped and analysed as indicated for the protein-primed initiation assay. The TP-dAMP formed in the different competition conditions relative to that formed in the absence of competition (100%) is indicated. As control of competition, the inactive TP mutant S232C was used. Data are represented as mean ± SD corresponding to three independent experiments.
Mentions: ϕ29 DNA replication starts from both origins placed at the termini of the linear ϕ29 TP-DNA molecule. The DNA polymerase forms a heterodimer with a free TP molecule and catalyses the template-directed insertion of 5′ dAMP onto the hydroxyl group of TP Ser232. To study the primer function of mutant TPs, the formation of the TP-dAMP complex (initiation reaction) was evaluated using as template ϕ29 TP-DNA. As shown in Figure 2A and Table 1, there were mutants severely (R158A) or moderately (R169A, Q253A and R256A) impaired in the formation of the TP-dAMP product. As the initiation of ϕ29 TP-DNA replication is a template-directed event, such deficiency could be a consequence of a faulty recognition/binding of the replication origins. To study this possibility, we made use of the ability exhibited by ϕ29 DNA polymerase to catalyse the deoxynucleotidylation of the TP in the absence of template, the formation of the TP-dAMP product relying exclusively on a functional DNA polymerase-TP interaction (24). As shown in Table 1, except for mutant D198A, the rest of the TP derivatives showed a drop in their priming ability. Thus, no activity was detected with mutants R158A, R169A, E252A, Q253A and R256A, and TP derivatives E191A and Y250A showed a decrease of 3- and 7-fold, respectively, in their priming activity relative to the template-directed reaction. Thus, the low TP-deoxynucleotidylation levels exhibited by TP mutants could be pointing to a defective interaction with the DNA polymerase.Figure 2.

Bottom Line: The 3D structure of the DNA polymerase/TP heterodimer allowed the identification of TP residues that could be responsible for interaction with the DNA polymerase.Here, we examined the role of TP residues Arg158, Arg169, Glu191, Asp198, Tyr250, Glu252, Gln253 and Arg256 by in vitro analyses of mutant derivatives.The results showed that substitution of these residues had an effect on either the stability of the TP/DNA polymerase complex (R158A) or in the functional interaction of the TP at the polymerization active site (R169A, E191A, Y250A, E252A, Q253A and R256A), affecting the first steps of Φ29 TP-DNA replication.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular Eladio Viñuela (CSIC), Centro de Biología Molecular Severo Ochoa (CSIC-UAM), C/Nicolás Cabrera 1, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.

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

Show MeSH
Related in: MedlinePlus