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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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Effect of p6 on the artificial replication origins. Initiation reactions were performed as described (see Materials and Methods section) but having an initial nucleotide concentration of 0.1 μM. The double-stranded DNA origins were constructed with oligonucleotides 68-nucleotides long, containing a p6 nucleation site, but the relevant ends were the same as those in the corresponding 29 bp origins. Where indicated, protein p6 was included in the reaction at a concentration of 35 μM. Quantification of the initiation bands are normalized in relation to the wt band (wt = 1); the corresponding standard deviations are shown. Stimulation factors by p6 are indicated for each origin.
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Figure 7: Effect of p6 on the artificial replication origins. Initiation reactions were performed as described (see Materials and Methods section) but having an initial nucleotide concentration of 0.1 μM. The double-stranded DNA origins were constructed with oligonucleotides 68-nucleotides long, containing a p6 nucleation site, but the relevant ends were the same as those in the corresponding 29 bp origins. Where indicated, protein p6 was included in the reaction at a concentration of 35 μM. Quantification of the initiation bands are normalized in relation to the wt band (wt = 1); the corresponding standard deviations are shown. Stimulation factors by p6 are indicated for each origin.

Mentions: As mentioned in the Introduction, two non-excluding mechanisms have been proposed to explain the p6 role in the stimulation of replication: to melt the DNA strands at the very ends of the phage genome and to recruit the TP/DNA polymerase preinitiation complex to the origin of replication. Given that some of the artificial origins described here have their ends already unpaired we considered they could serve as a test to unravel the p6 mechanism. The effect of p6 is mostly evident in the initiation step with a moderately low nucleotide concentration (0.1 μM), so we performed an initiation assay (see Materials and Methods section) using 68 bp double-stranded origins containing the high affinity p6 nucleation sites localized between positions 35 and 58 (17), and the most relevant end sequences and structures described in this study. In Figure 7, the p6-induced factors of stimulation were 2.8 for the wt origin, 3.6 for GC and 1.6, 1.3, 1.9, 1.4 and 0.8 for the A/C, 6m, 8m, 10m and 12m origins, respectively. It is observed that the effect of p6 is lower with the unpaired origins down to no effect with 12 mismatches. This lower level of stimulation by p6 would suggest that, at least in part, the unpaired origins are favoring the same step of replication as p6. And this in turn is in agreement with the explanation that the stimulation by p6 and the effects of the unpaired origins act through the melting of the dsDNA at the origin. Notwithstanding, an alternative explanation would be that the unpairings would dissipate the torsional strength generated by the p6 complex therefore affecting its function. In any case, with the exception of the 12m origin, there is always a remaining level of stimulation by p6 that could be attributed to a different p6 action-mechanism like the direct interaction of p6 with the complex TP/DNA polymerase, as it has been previously suggested (20,21).


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)

Effect of p6 on the artificial replication origins. Initiation reactions were performed as described (see Materials and Methods section) but having an initial nucleotide concentration of 0.1 μM. The double-stranded DNA origins were constructed with oligonucleotides 68-nucleotides long, containing a p6 nucleation site, but the relevant ends were the same as those in the corresponding 29 bp origins. Where indicated, protein p6 was included in the reaction at a concentration of 35 μM. Quantification of the initiation bands are normalized in relation to the wt band (wt = 1); the corresponding standard deviations are shown. Stimulation factors by p6 are indicated for each origin.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Effect of p6 on the artificial replication origins. Initiation reactions were performed as described (see Materials and Methods section) but having an initial nucleotide concentration of 0.1 μM. The double-stranded DNA origins were constructed with oligonucleotides 68-nucleotides long, containing a p6 nucleation site, but the relevant ends were the same as those in the corresponding 29 bp origins. Where indicated, protein p6 was included in the reaction at a concentration of 35 μM. Quantification of the initiation bands are normalized in relation to the wt band (wt = 1); the corresponding standard deviations are shown. Stimulation factors by p6 are indicated for each origin.
Mentions: As mentioned in the Introduction, two non-excluding mechanisms have been proposed to explain the p6 role in the stimulation of replication: to melt the DNA strands at the very ends of the phage genome and to recruit the TP/DNA polymerase preinitiation complex to the origin of replication. Given that some of the artificial origins described here have their ends already unpaired we considered they could serve as a test to unravel the p6 mechanism. The effect of p6 is mostly evident in the initiation step with a moderately low nucleotide concentration (0.1 μM), so we performed an initiation assay (see Materials and Methods section) using 68 bp double-stranded origins containing the high affinity p6 nucleation sites localized between positions 35 and 58 (17), and the most relevant end sequences and structures described in this study. In Figure 7, the p6-induced factors of stimulation were 2.8 for the wt origin, 3.6 for GC and 1.6, 1.3, 1.9, 1.4 and 0.8 for the A/C, 6m, 8m, 10m and 12m origins, respectively. It is observed that the effect of p6 is lower with the unpaired origins down to no effect with 12 mismatches. This lower level of stimulation by p6 would suggest that, at least in part, the unpaired origins are favoring the same step of replication as p6. And this in turn is in agreement with the explanation that the stimulation by p6 and the effects of the unpaired origins act through the melting of the dsDNA at the origin. Notwithstanding, an alternative explanation would be that the unpairings would dissipate the torsional strength generated by the p6 complex therefore affecting its function. In any case, with the exception of the 12m origin, there is always a remaining level of stimulation by p6 that could be attributed to a different p6 action-mechanism like the direct interaction of p6 with the complex TP/DNA polymerase, as it has been previously suggested (20,21).

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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