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: From previous work it was known that the replication machinery can utilize ends with 3′ protrusions 5-nucleotides long, because the intrinsic 3′ to 5′ exonuclease activity of the Φ29 DNA polymerase can eliminate the extensions to generate the wild-type, blunt ends (34). Since the unpairing of the ends has a positive effect on origin utilization, we tested the effect of an extended displaced strand on the origin strength with the idea that an increased number of interactions between a non-template protruding single strand and the TP/DNA polymerase heterodimer could stabilize the initiation complex and produce higher origin utilization. Thus, we tested origins with different 5′ 10-nucleotides ssDNA overhangs at the displaced strand, taking into account that these overhangs cannot be degraded by the 3′-5′ exonuclease activity of the Φ29 DNA polymerase. The overhangs were 10-nucleotides long in order to provide enough ssDNA to cover a substantial portion of the TP/DNA polymerase heterodimer. Since a 3′-recessive DNA end would be a good primer for the standard polymerization activity, and this would alter the ends to analyze, we constructed non-extensible oligonucleotides with dideoxy-CMP (ddC) as the last nucleotide at the 3′ position of the template strand. As a control for the possible additional effect of the templates with ddC, we tested the two constructions A/C and G/C with and without ddC. In Figure 2 we observe that both A/C and G/C origins with ddC (lanes A/ddC and G/ddC, respectively) at the very end have 25% of the activity of their homologous origins with dC at the end. Although the reason for this is unknown, a possibility is that the 3′ hydroxyl group may be important in the interaction with the TP at the early event of initiation. As an additional fact, we also observed that the replication activity from a double-stranded origin (wtds) was stronger than that of a single-stranded origin (wtss), having just the template strand. This stresses the importance of the displaced strand in the early process of replication. Regarding the 5′-overhang origins we observed that they all lead to markedly lower activities than the A/ddC non-overhang control. It happens so if the origin has the 5′ protruding sequence ATCACTATGC and then AAA/ddCCC, or the same 5′ extension and then TAC/ddCCC (lanes 10+A/ddC and 10+TAC/ddC), or even if it has the 5′-overhang sequence AAACTATGC and then TAC/ddCCC (lane AAA10); in all cases the activity was lower than that of the A/ddC and the wild-type origins. As expected, the overhang with G/C pairing at the end of the double-stranded section showed an activity even lower than that of the other origins (lane 10+G/ddC). Also, a wild-type origin with a 5′-overhang that forms a self-complementary hairpin (lane 5′hA/T) and other modified wild-type origin lacking six nucleotides of the displaced strand (lane 5′Δ6wt) showed essentially no activity, probably because the heterodimer has to face the opening of a G/C paired double-stranded DNA at the same time as initiation is taking place. These results point to the importance of the displaced strand in the process of origin utilization, and they also suggest that, when the displaced strand is made artificially longer than the template, it does not produce stimulation, contrary to what happens when we simulate the effect of an origin being opened by using unpaired oligonucleotides of the same length.
Affiliation: Centro de Biología Molecular "Severo Ochoa" (Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.