Improved artificial origins for phage Φ29 terminal protein-primed replication. Insights into early replication events.
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.
Affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.Show MeSH
Mentions: Since the artificial origin with six unpaired nucleotides (6m) at the end was the strongest of the combinations tested, we wondered whether longer unpaired regions could have even stronger positive effects on origin utilization. For that purpose, we generated origins with a total of 8, 10 or 12 unpaired nucleotides at the end. To eliminate as much as possible the effect of sequence, we designed origins sharing the template strand sequence and the six first nucleotides of the displaced strand. So, for example, the construction 8m has eight unpairings, of which, the first six were the same as that of construction 6m but the nucleotides at positions 7 and 8 are different on the displaced strand so they generate mismatches. Also, in this experiment, to avoid melting effects due to unpairing of substantial portions of 29mer oligonucleotides, we utilized 68mer oligonucleotides since, for the 68mers, the melting effect of the unpaired portions is negligible at the temperature tested. A control experiment was performed to compare the replication activities of 29mer and 68mer templates (see Supplementary Figure S2). The results (Figure 4) show that the optimum unpairing is six nucleotides since this construct has the highest activity, 17.4-fold that of the wild-type (lane 6m). This maximal activity is lower (about 2-fold) than that obtained with 29mer oligonucleotides, and we presently do not know the reason for this. As further unpairing is added, the activity keeps getting reduced down to 2.4-fold the wild-type activity for 12 mismatched positions (lane 12m).
Affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.