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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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Analysis of TP/DNA polymerase interaction by glycerol gradient ultracentrifugation. The assay was carried out as described under ‘Materials and Methods’ section, pre-incubating 3 µg of either wild-type or the indicated mutant TP with 6 µg of DNA polymerase. After incubation for 30 min at 4°C, samples were loaded on top of a continuous 15–30% glycerol gradient in the presence of 0.2 M NaCl. After centrifugation, the collected fractions were subjected to SDS–12% PAGE and further stained with SYPRO. Densitometric quantification, expressed in arbitrary units, of both DNA polymerase (full circles) and TP (open circles) are represented.
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gkr1283-F3: Analysis of TP/DNA polymerase interaction by glycerol gradient ultracentrifugation. The assay was carried out as described under ‘Materials and Methods’ section, pre-incubating 3 µg of either wild-type or the indicated mutant TP with 6 µg of DNA polymerase. After incubation for 30 min at 4°C, samples were loaded on top of a continuous 15–30% glycerol gradient in the presence of 0.2 M NaCl. After centrifugation, the collected fractions were subjected to SDS–12% PAGE and further stained with SYPRO. Densitometric quantification, expressed in arbitrary units, of both DNA polymerase (full circles) and TP (open circles) are represented.

Mentions: The interaction of mutant TPs with the DNA polymerase was also directly analysed by glycerol gradient ultracentrifugation in the presence of 0.2 M NaCl (see Materials and Methods). As it can be observed in Figure 3, the wild-type TP and the mutant derivatives, with the sole exception of mutant R158A, formed a heterodimer with the DNA polymerase. Thus, although it seemed that the stability of the heterodimer was not hindered by the mutations introduced, the poor deoxynucleotidylation activity exhibited by the mutant TPs in the absence of template, as well as the lack of competition with the wild-type TP (see above), would be reflecting a non-functional interaction with the DNA polymerase. To further analyse the stability of the DNA polymerase/TP complexes, glycerol gradient centrifugation analysis was carried out in the presence of 0.4 M NaCl (Supplementary Figure 1). Under these conditions, whereas the wild-type and mutant TPs E191A and D198A still formed a stable heterodimer with the DNA polymerase, mutants R158A, R169A, Y250A, E252A, Q253A and R256A eluted separately, showing that their interaction with the DNA polymerase is not as strong as that displayed by the wild-type DNA polymerase.Figure 3.


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)

Analysis of TP/DNA polymerase interaction by glycerol gradient ultracentrifugation. The assay was carried out as described under ‘Materials and Methods’ section, pre-incubating 3 µg of either wild-type or the indicated mutant TP with 6 µg of DNA polymerase. After incubation for 30 min at 4°C, samples were loaded on top of a continuous 15–30% glycerol gradient in the presence of 0.2 M NaCl. After centrifugation, the collected fractions were subjected to SDS–12% PAGE and further stained with SYPRO. Densitometric quantification, expressed in arbitrary units, of both DNA polymerase (full circles) and TP (open circles) are represented.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351185&req=5

gkr1283-F3: Analysis of TP/DNA polymerase interaction by glycerol gradient ultracentrifugation. The assay was carried out as described under ‘Materials and Methods’ section, pre-incubating 3 µg of either wild-type or the indicated mutant TP with 6 µg of DNA polymerase. After incubation for 30 min at 4°C, samples were loaded on top of a continuous 15–30% glycerol gradient in the presence of 0.2 M NaCl. After centrifugation, the collected fractions were subjected to SDS–12% PAGE and further stained with SYPRO. Densitometric quantification, expressed in arbitrary units, of both DNA polymerase (full circles) and TP (open circles) are represented.
Mentions: The interaction of mutant TPs with the DNA polymerase was also directly analysed by glycerol gradient ultracentrifugation in the presence of 0.2 M NaCl (see Materials and Methods). As it can be observed in Figure 3, the wild-type TP and the mutant derivatives, with the sole exception of mutant R158A, formed a heterodimer with the DNA polymerase. Thus, although it seemed that the stability of the heterodimer was not hindered by the mutations introduced, the poor deoxynucleotidylation activity exhibited by the mutant TPs in the absence of template, as well as the lack of competition with the wild-type TP (see above), would be reflecting a non-functional interaction with the DNA polymerase. To further analyse the stability of the DNA polymerase/TP complexes, glycerol gradient centrifugation analysis was carried out in the presence of 0.4 M NaCl (Supplementary Figure 1). Under these conditions, whereas the wild-type and mutant TPs E191A and D198A still formed a stable heterodimer with the DNA polymerase, mutants R158A, R169A, Y250A, E252A, Q253A and R256A eluted separately, showing that their interaction with the DNA polymerase is not as strong as that displayed by the wild-type DNA polymerase.Figure 3.

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