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Improved artificial origins for phage Φ29 terminal protein-primed replication. Insights into early replication events.

Gella P, Salas M, Mencía M - Nucleic Acids Res. (2014)

Bottom Line: To better understand the early replication events and to find improved origins for DNA amplification based on the Φ29 system, we have studied the end-structure of a double-stranded DNA replication origin.We also show that the presence of a correctly positioned displaced strand is important because origins with 5' or 3' ssDNA regions have very low activity.We suggest that the template and non-template strands of the origin and the TP/DNA polymerase complex form series of interactions that control the critical start of terminal protein-primed replication.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.

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Role of the TP Nt domain in replication using artificial replication origins. Notation of origins as described before. AAA6m’ denotes AAAGTA/CCCGTA and CAA6m’ denotes CAAGTA/CCCGTA. The replication reactions were performed in the presence of 150 nM of either wild-type TP (TPwt) or the ΔNt deletion mutant of TP (TPΔNt). M, marker for the initiation band for the corresponding TP.
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Figure 6: Role of the TP Nt domain in replication using artificial replication origins. Notation of origins as described before. AAA6m’ denotes AAAGTA/CCCGTA and CAA6m’ denotes CAAGTA/CCCGTA. The replication reactions were performed in the presence of 150 nM of either wild-type TP (TPwt) or the ΔNt deletion mutant of TP (TPΔNt). M, marker for the initiation band for the corresponding TP.

Mentions: It has been reported that the TP N-terminal (Nt) domain has non-sequence specific DNA binding capacity and also that it is partially dispensable for in vitro replication of Φ29 DNA (21). To understand the role of the Nt domain in the first steps of replication, we tested a TP mutant that lacks the Nt domain (TPΔNt) in replication assays with different double-stranded origins. As shown before, the identity in the 5′ end of the displaced strand affected the reaction efficiency so we hypothesized that the Nt domain of the TP could have a recognition role of the 5′ end. Thus, we performed replication assays using the TPΔNt and different origins altered on the first 5′ nucleotide. In order to use the same non-template sequences as those in Figure 3, we constructed a six mismatched origin with the sequence AAAGTA/CCCGTA (AAA6m’), and the mutant lacking one A at the 5′ end CAAGTA/CCCGTA (CAA6m’). As shown in Figure 6 (lanes wt), replication utilizing the wild-type origin as template was very much reduced with the TPΔNt, about 30-fold compared to that of the wild-type TP. Similarly, the origins A/C and CAA (both with three mismatches) showed very low utilization (lanes A/C and CAA) in the presence of the TPΔNt. However, the six mismatched constructs showed strong activity in the presence of TPΔNt (lanes 6m and AAA 6m’) and this activity was clearly dependent on A at the 5′ end (lane CAA 6m’). Therefore, origin utilization by the TPΔNt complex has a preference for A at the 5′ position in the six mismatched construct. At the same time, the wild-type TP is hardly affected by the change of the 5′ A into C in the 6m origin (lane CAA 6m’).


Improved artificial origins for phage Φ29 terminal protein-primed replication. Insights into early replication events.

Gella P, Salas M, Mencía M - Nucleic Acids Res. (2014)

Role of the TP Nt domain in replication using artificial replication origins. Notation of origins as described before. AAA6m’ denotes AAAGTA/CCCGTA and CAA6m’ denotes CAAGTA/CCCGTA. The replication reactions were performed in the presence of 150 nM of either wild-type TP (TPwt) or the ΔNt deletion mutant of TP (TPΔNt). M, marker for the initiation band for the corresponding TP.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150772&req=5

Figure 6: Role of the TP Nt domain in replication using artificial replication origins. Notation of origins as described before. AAA6m’ denotes AAAGTA/CCCGTA and CAA6m’ denotes CAAGTA/CCCGTA. The replication reactions were performed in the presence of 150 nM of either wild-type TP (TPwt) or the ΔNt deletion mutant of TP (TPΔNt). M, marker for the initiation band for the corresponding TP.
Mentions: It has been reported that the TP N-terminal (Nt) domain has non-sequence specific DNA binding capacity and also that it is partially dispensable for in vitro replication of Φ29 DNA (21). To understand the role of the Nt domain in the first steps of replication, we tested a TP mutant that lacks the Nt domain (TPΔNt) in replication assays with different double-stranded origins. As shown before, the identity in the 5′ end of the displaced strand affected the reaction efficiency so we hypothesized that the Nt domain of the TP could have a recognition role of the 5′ end. Thus, we performed replication assays using the TPΔNt and different origins altered on the first 5′ nucleotide. In order to use the same non-template sequences as those in Figure 3, we constructed a six mismatched origin with the sequence AAAGTA/CCCGTA (AAA6m’), and the mutant lacking one A at the 5′ end CAAGTA/CCCGTA (CAA6m’). As shown in Figure 6 (lanes wt), replication utilizing the wild-type origin as template was very much reduced with the TPΔNt, about 30-fold compared to that of the wild-type TP. Similarly, the origins A/C and CAA (both with three mismatches) showed very low utilization (lanes A/C and CAA) in the presence of the TPΔNt. However, the six mismatched constructs showed strong activity in the presence of TPΔNt (lanes 6m and AAA 6m’) and this activity was clearly dependent on A at the 5′ end (lane CAA 6m’). Therefore, origin utilization by the TPΔNt complex has a preference for A at the 5′ position in the six mismatched construct. At the same time, the wild-type TP is hardly affected by the change of the 5′ A into C in the 6m origin (lane CAA 6m’).

Bottom Line: To better understand the early replication events and to find improved origins for DNA amplification based on the Φ29 system, we have studied the end-structure of a double-stranded DNA replication origin.We also show that the presence of a correctly positioned displaced strand is important because origins with 5' or 3' ssDNA regions have very low activity.We suggest that the template and non-template strands of the origin and the TP/DNA polymerase complex form series of interactions that control the critical start of terminal protein-primed replication.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.

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