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: As mentioned in the Introduction, phage Φ29 DNA replication starts with an initiation step, up to the incorporation of the third nucleotide and then follows a transition step until the nascent strand has 6–9 nucleotides. At that point some change takes place and the DNA polymerase switches to the elongation mode from the 10th position onwards after the DNA polymerase and the TP have dissociated. Some of the replication origins described in previous sections were stronger than the wild-type origin, especially those with the AAA/CCC structure at the very end. In order to analyze what step or steps of the replication process are being facilitated in these artificial origins we performed transition assays (see Materials and Methods) with the A/C and 6m origins (Figure 5). In these truncated elongation experiments the reactions included: lanes a, only the first nucleotide to be incorporated (to produce AAA or GGGG depending on the template; initiation bands); lanes b, the first nucleotide (A or G) plus ddG (to yield AAAG in the wild-type origin) or plus ddT (to yield GGGGT in the A/C and 6m origins); lanes c, nucleotides A, G plus ddT (to produce AAAGT in the wild-type origin) or nucleotides G, T plus ddA (to produce GGGGTA in the A/C and 6m origins); lanes d, nucleotides A, G and T plus ddC (that allow polymerization of 12 nucleotides in all constructs) and lanes e, the four nucleotides for full replication of the template. We observed that, for the wild-type origin, the initiation bands (lane a) progressed with some efficiency to position +4 (lane b), but with much less efficiency to positions +5, +12 or full length (lanes c, d, e), taking into account the number of labeled nucleotides that can be incorporated for each length of synthesized DNA. For the A/C and 6m templates we observed an increase in the initiation bands of 2.3 to 2.4 times with respect to those of the wild-type origin but also a further and stronger increase when the replication proceeds to position +12 (11-fold, A/C, or 21-fold, 6m, increase) or full length. The results suggest that the A/C sequences plus the unpairings enhance the initiation step but they also strongly stimulate the passage from transition to elongation.
Affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.