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: The best activities so far were obtained with origins with the unpairing A/C at the end. For that reason, we decided to characterize the observed preference for the sequence AAA at the 5′ end of the displaced strand. We started from the original 3-mismatched A/C origin (A/C = AAA in Figure 3), because it shows a clearer effect of the AAA sequence than other origins. We substituted all As at the displaced strand individually or in combinations with Cs. The results (Figure 3) showed that there is a strong preference for A at the first position from the 5′ end (compare lane AAA with CAA). The substitution of the second A also reduces the activity of the origin (lanes AAA versus ACA and lanes CAC versus CCC), although the effect is lower than that of the first A. However, for position 3 there are cases where its substitution reduces the activity (lanes AAA versus AAC and lanes ACA versus ACC) and other cases where the change increases slightly the activity of the origin (lanes CAA versus CAC and CCA versus CCC). Comparing the origins with just one A (ACC, CAC and CCA) a possible ranking of importance can be suggested, first A better than second A and this is better than the third A. The results suggest that there is a preference for AAA at the 5′ end of the displaced strand by the TP/DNA polymerase complex and that the first A is the most important one for that preference.
Affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.