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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) TP-DNA replication by mutant TPs. The assays were carried as described in ‘Materials and Methods’ section in the presence of 20 ng of DNA polymerase and 10 ng of either wild-type or the indicated mutant TP. After incubation at the indicated times at 30°C, relative values were calculated (Table 1) and the length of the synthesized DNA was analysed by alkaline agarose gel electrophoresis. The migration position of unit-length ϕ29 DNA is indicated. (B) Analysis of the transition products of ϕ29 DNA replication. The assay was performed as described in ‘Materials and Methods’ section in the presence of 20 ng of DNA polymerase mutant N62D (22), 10 ng of either wild-type or the indicated mutant TP and 5 µM of each dATP, dGTP and dTTP. After incubation for 2 min at 30°C, the different transition products were detected and analysed by high resolution SDS–PAGE. The products expected during the first replication steps, starting from both ends of ϕ29 TP-DNA [(TP-dAMP, TP-(dAMP)2, TP-(dNMP)8 and TP-(dNMP11)], as well as TP-(dNMP)6 and TP-(dNMP)14, are indicated.
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gkr1283-F4: (A) TP-DNA replication by mutant TPs. The assays were carried as described in ‘Materials and Methods’ section in the presence of 20 ng of DNA polymerase and 10 ng of either wild-type or the indicated mutant TP. After incubation at the indicated times at 30°C, relative values were calculated (Table 1) and the length of the synthesized DNA was analysed by alkaline agarose gel electrophoresis. The migration position of unit-length ϕ29 DNA is indicated. (B) Analysis of the transition products of ϕ29 DNA replication. The assay was performed as described in ‘Materials and Methods’ section in the presence of 20 ng of DNA polymerase mutant N62D (22), 10 ng of either wild-type or the indicated mutant TP and 5 µM of each dATP, dGTP and dTTP. After incubation for 2 min at 30°C, the different transition products were detected and analysed by high resolution SDS–PAGE. The products expected during the first replication steps, starting from both ends of ϕ29 TP-DNA [(TP-dAMP, TP-(dAMP)2, TP-(dNMP)8 and TP-(dNMP11)], as well as TP-(dNMP)6 and TP-(dNMP)14, are indicated.

Mentions: Once it catalyses the formation of the TP-dAMP product, the same DNA polymerase molecule elongates it via strand displacement to produce full-length ϕ29 TP-DNA. To ascertain to what extent the impaired DNA polymerase/mutant TP interaction affects the replication process, replication assays were carried out, using a minimal replication system based on ϕ29 TP-DNA, DNA polymerase and TP (14) (see ‘Materials and Methods’ section). As observed in Figure 4A and Table 1, when mutant R158A was used as primer TP, the efficiency of the reaction decreased 6-fold respect to the wild-type TP, in accordance with its poor priming proficiency. Nonetheless, when the rest of mutant TPs were used as primer, the efficiency of the reaction was either not affected (TP mutants E191A, Y250A, E252A) or only slightly reduced (TP mutants R169A, D198A, Q253A and R256A), regardless of their impaired interaction with the DNA polymerase. These results could suggest that formation of a catalytically competent heterodimer depends on contacts with the parental TP that could alleviate at least partially the defects caused by the mutations introduced in the TP.Figure 4.


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) TP-DNA replication by mutant TPs. The assays were carried as described in ‘Materials and Methods’ section in the presence of 20 ng of DNA polymerase and 10 ng of either wild-type or the indicated mutant TP. After incubation at the indicated times at 30°C, relative values were calculated (Table 1) and the length of the synthesized DNA was analysed by alkaline agarose gel electrophoresis. The migration position of unit-length ϕ29 DNA is indicated. (B) Analysis of the transition products of ϕ29 DNA replication. The assay was performed as described in ‘Materials and Methods’ section in the presence of 20 ng of DNA polymerase mutant N62D (22), 10 ng of either wild-type or the indicated mutant TP and 5 µM of each dATP, dGTP and dTTP. After incubation for 2 min at 30°C, the different transition products were detected and analysed by high resolution SDS–PAGE. The products expected during the first replication steps, starting from both ends of ϕ29 TP-DNA [(TP-dAMP, TP-(dAMP)2, TP-(dNMP)8 and TP-(dNMP11)], as well as TP-(dNMP)6 and TP-(dNMP)14, are indicated.
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gkr1283-F4: (A) TP-DNA replication by mutant TPs. The assays were carried as described in ‘Materials and Methods’ section in the presence of 20 ng of DNA polymerase and 10 ng of either wild-type or the indicated mutant TP. After incubation at the indicated times at 30°C, relative values were calculated (Table 1) and the length of the synthesized DNA was analysed by alkaline agarose gel electrophoresis. The migration position of unit-length ϕ29 DNA is indicated. (B) Analysis of the transition products of ϕ29 DNA replication. The assay was performed as described in ‘Materials and Methods’ section in the presence of 20 ng of DNA polymerase mutant N62D (22), 10 ng of either wild-type or the indicated mutant TP and 5 µM of each dATP, dGTP and dTTP. After incubation for 2 min at 30°C, the different transition products were detected and analysed by high resolution SDS–PAGE. The products expected during the first replication steps, starting from both ends of ϕ29 TP-DNA [(TP-dAMP, TP-(dAMP)2, TP-(dNMP)8 and TP-(dNMP11)], as well as TP-(dNMP)6 and TP-(dNMP)14, are indicated.
Mentions: Once it catalyses the formation of the TP-dAMP product, the same DNA polymerase molecule elongates it via strand displacement to produce full-length ϕ29 TP-DNA. To ascertain to what extent the impaired DNA polymerase/mutant TP interaction affects the replication process, replication assays were carried out, using a minimal replication system based on ϕ29 TP-DNA, DNA polymerase and TP (14) (see ‘Materials and Methods’ section). As observed in Figure 4A and Table 1, when mutant R158A was used as primer TP, the efficiency of the reaction decreased 6-fold respect to the wild-type TP, in accordance with its poor priming proficiency. Nonetheless, when the rest of mutant TPs were used as primer, the efficiency of the reaction was either not affected (TP mutants E191A, Y250A, E252A) or only slightly reduced (TP mutants R169A, D198A, Q253A and R256A), regardless of their impaired interaction with the DNA polymerase. These results could suggest that formation of a catalytically competent heterodimer depends on contacts with the parental TP that could alleviate at least partially the defects caused by the mutations introduced in the TP.Figure 4.

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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