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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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In vitro ϕ29 TP-DNA amplification with TP mutant R169A is recovered upon addition of wild-type TP/DNA heterodimer. (A) Amplification assays were performed using the conditions described under ‘Materials and Methods’ section. After 80 min, TP/DNA polymerase heterodimers containing either wild-type or the TP mutant R169A were added to the reaction mixtures. The samples, withdrawn at the indicated times, were analysed as described in the legend of Figure 6. The amount of incorporated [α-32P]dAMP was measured at the indicated times and is represented in (B). White bars, amount of incorporated [α-32P]dAMP by mutant TP/DNA polymerase throughout the experiment. Black bars, amount of newly incorporated [α-32P]dAMP upon addition of wild-type TP/DNA polymerase heterodimers. Grey bars, amount of newly incorporated [α-32P]dAMP upon addition of mutant TP/DNA polymerase heterodimers after 80 min of reaction. Data are represented as mean ± SD corresponding to four independent experiments.
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gkr1283-F7: In vitro ϕ29 TP-DNA amplification with TP mutant R169A is recovered upon addition of wild-type TP/DNA heterodimer. (A) Amplification assays were performed using the conditions described under ‘Materials and Methods’ section. After 80 min, TP/DNA polymerase heterodimers containing either wild-type or the TP mutant R169A were added to the reaction mixtures. The samples, withdrawn at the indicated times, were analysed as described in the legend of Figure 6. The amount of incorporated [α-32P]dAMP was measured at the indicated times and is represented in (B). White bars, amount of incorporated [α-32P]dAMP by mutant TP/DNA polymerase throughout the experiment. Black bars, amount of newly incorporated [α-32P]dAMP upon addition of wild-type TP/DNA polymerase heterodimers. Grey bars, amount of newly incorporated [α-32P]dAMP upon addition of mutant TP/DNA polymerase heterodimers after 80 min of reaction. Data are represented as mean ± SD corresponding to four independent experiments.

Mentions: In contrast with in vitro replication, performed in the presence of the DNA polymerase and the TP, the addition to the reaction of DBP and SSB enables 103-fold amplification of limiting amounts (0.5 ng) of input ϕ29 TP-DNA (23). Figure 5 shows the amount of DNA amplified using wild-type or mutant TPs. Mutants E191A, D198A and Y250A led to amplification levels similar or even higher than those reached with the wild-type TP (Table 1), and the amplification yield reached with mutant E252A was only 2-fold lower than that obtained with the wild-type TP. Conversely, mutants R158A, R169A, Q253A and R256A were inefficient in supporting the amplification reaction most likely because of their deficient priming activity (Figure 5 and Table 1). In these latter cases, the amount of de novo synthesized DNA (in nanograms) was quantified from the amount of radioactivity incorporated into DNA as a function of time (Figure 6, upper panel), the amplification factor being calculated as the ratio between the amount of DNA at the end of the reaction (input DNA plus synthesized DNA) and the amount of input DNA (Figure 6, lower panel). As shown in Figure 6, with the wild-type TP, DNA synthesis reaches a plateau after 40 min giving rise to an amplification factor of 50. Whereas the DNA polymerase did not synthesize any detectable product when using TP mutant R158A, DNA synthesis with mutants R256A and Q253A did not reach a plateau at the longest time assayed (160 min), suggesting that these mutations slow down the starting of new replication rounds. Interestingly, when TP mutant R169A was used as primer, although synthesis reached a plateau after 80 min, the amount of de novo synthesized DNA corresponded to an amplification factor of 3 (Figure 6). This result suggests that DNA synthesis has stalled before completing a second round of replication, possibly due to the inactivation of the replication origin upon incorporation of the mutant TP that now would act as parental TP. If this were the case, only the wild-type TP-containing origins would be active in the second round of replication resulting in a maximum amplification factor of 3, as previously described for other TP mutants (27). Interestingly, renewed incorporation of dAMP was observed upon the addition of wild-type TP/DNA polymerase heterodimer after 80 min of reaction (Figure 7) ruling out the possibility that the mutation had affected the overall structure of the parental TP that might cause inactivation of the origins. As expected, addition of mutant TP/DNA polymerase heterodimer did not result in renewed dAMP incorporation. These results suggest that the parental TP mutant R169A could produce functional interactions with wild-type TP/DNA polymerase heterodimer but not with a heterodimer containing the mutant TP. Therefore, the mutation is critical when it is present in both the primer and parental TP, presumably affecting their proper interaction required for the initiation reaction.Figure 5.


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)

In vitro ϕ29 TP-DNA amplification with TP mutant R169A is recovered upon addition of wild-type TP/DNA heterodimer. (A) Amplification assays were performed using the conditions described under ‘Materials and Methods’ section. After 80 min, TP/DNA polymerase heterodimers containing either wild-type or the TP mutant R169A were added to the reaction mixtures. The samples, withdrawn at the indicated times, were analysed as described in the legend of Figure 6. The amount of incorporated [α-32P]dAMP was measured at the indicated times and is represented in (B). White bars, amount of incorporated [α-32P]dAMP by mutant TP/DNA polymerase throughout the experiment. Black bars, amount of newly incorporated [α-32P]dAMP upon addition of wild-type TP/DNA polymerase heterodimers. Grey bars, amount of newly incorporated [α-32P]dAMP upon addition of mutant TP/DNA polymerase heterodimers after 80 min of reaction. Data are represented as mean ± SD corresponding to four independent experiments.
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Related In: Results  -  Collection

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gkr1283-F7: In vitro ϕ29 TP-DNA amplification with TP mutant R169A is recovered upon addition of wild-type TP/DNA heterodimer. (A) Amplification assays were performed using the conditions described under ‘Materials and Methods’ section. After 80 min, TP/DNA polymerase heterodimers containing either wild-type or the TP mutant R169A were added to the reaction mixtures. The samples, withdrawn at the indicated times, were analysed as described in the legend of Figure 6. The amount of incorporated [α-32P]dAMP was measured at the indicated times and is represented in (B). White bars, amount of incorporated [α-32P]dAMP by mutant TP/DNA polymerase throughout the experiment. Black bars, amount of newly incorporated [α-32P]dAMP upon addition of wild-type TP/DNA polymerase heterodimers. Grey bars, amount of newly incorporated [α-32P]dAMP upon addition of mutant TP/DNA polymerase heterodimers after 80 min of reaction. Data are represented as mean ± SD corresponding to four independent experiments.
Mentions: In contrast with in vitro replication, performed in the presence of the DNA polymerase and the TP, the addition to the reaction of DBP and SSB enables 103-fold amplification of limiting amounts (0.5 ng) of input ϕ29 TP-DNA (23). Figure 5 shows the amount of DNA amplified using wild-type or mutant TPs. Mutants E191A, D198A and Y250A led to amplification levels similar or even higher than those reached with the wild-type TP (Table 1), and the amplification yield reached with mutant E252A was only 2-fold lower than that obtained with the wild-type TP. Conversely, mutants R158A, R169A, Q253A and R256A were inefficient in supporting the amplification reaction most likely because of their deficient priming activity (Figure 5 and Table 1). In these latter cases, the amount of de novo synthesized DNA (in nanograms) was quantified from the amount of radioactivity incorporated into DNA as a function of time (Figure 6, upper panel), the amplification factor being calculated as the ratio between the amount of DNA at the end of the reaction (input DNA plus synthesized DNA) and the amount of input DNA (Figure 6, lower panel). As shown in Figure 6, with the wild-type TP, DNA synthesis reaches a plateau after 40 min giving rise to an amplification factor of 50. Whereas the DNA polymerase did not synthesize any detectable product when using TP mutant R158A, DNA synthesis with mutants R256A and Q253A did not reach a plateau at the longest time assayed (160 min), suggesting that these mutations slow down the starting of new replication rounds. Interestingly, when TP mutant R169A was used as primer, although synthesis reached a plateau after 80 min, the amount of de novo synthesized DNA corresponded to an amplification factor of 3 (Figure 6). This result suggests that DNA synthesis has stalled before completing a second round of replication, possibly due to the inactivation of the replication origin upon incorporation of the mutant TP that now would act as parental TP. If this were the case, only the wild-type TP-containing origins would be active in the second round of replication resulting in a maximum amplification factor of 3, as previously described for other TP mutants (27). Interestingly, renewed incorporation of dAMP was observed upon the addition of wild-type TP/DNA polymerase heterodimer after 80 min of reaction (Figure 7) ruling out the possibility that the mutation had affected the overall structure of the parental TP that might cause inactivation of the origins. As expected, addition of mutant TP/DNA polymerase heterodimer did not result in renewed dAMP incorporation. These results suggest that the parental TP mutant R169A could produce functional interactions with wild-type TP/DNA polymerase heterodimer but not with a heterodimer containing the mutant TP. Therefore, the mutation is critical when it is present in both the primer and parental TP, presumably affecting their proper interaction required for the initiation reaction.Figure 5.

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