Improved artificial origins for phage Φ29 terminal protein-primed replication. Insights into early replication events.
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.
- DNA Replication*
- Replication Origin*
- Viral Proteins/chemistry/metabolism*
- Virus Replication*
- Bacillus Phages/genetics/physiology
- DNA-Binding Proteins/metabolism
- DNA-Directed DNA Polymerase/metabolism
- Protein Structure, Tertiary
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Figure 1: (A) Effect of group substitution of the first three nucleotides at the template and displaced strands on replication origin utilization. Double-stranded DNA origins were assembled with synthetic oligonucleotides to generate the different end combinations. Reactions were performed as described in Materials and Methods using [α32P] dATP as labeling nucleotide, and the reaction products were analyzed by high-resolution SDS-PAGE with 12% acrylamide. The positions corresponding to TP-dAMP (1) and full length product (TP-29 nucleotides ssDNA) are indicated. M, initiation reaction marker (reaction using TP-DNA as template and dATP as the only nucleotide, see Materials and Methods section); TP-DNA, control reaction with TP-containing Φ29 genome as template (this reaction produces the initiation and partial elongation bands [+15,+16] in addition to full-length Φ29 DNA [not shown]). The different pairs of oligonucleotides hybridized to generate the artificial origins are noted as follows (see scheme above Figure 1): the first letter stands for the first three nucleotides of the displaced strand and the second letter stands for the first three nucleotides of the template strand (for example, A/T denotes an origin with the paired sequence at the end AAA/TTT). The rest of the origin is the sequence of the Φ29 DNA right end from positions 4–29 (see Materials and Methods section). For example, the wt origin is the A/T combination. At the bottom of the figure are shown the mean values of the quantification of the intensities of the bands corresponding to full length products compared to the wt (wt = 1), with their corresponding standard deviations. Values are the average of at least three independent experiments. (B) Effect of nucleotide substitutions at positions 4–6 and unpairings on replication origin utilization. As previously, double-stranded DNAs were assembled with synthetic oligonucleotides to generate the different combinations. Reactions were analyzed by 12% SDS-PAGE. The positions corresponding to TP-dAMP (1) and full length product (TP-29 nucleotides ssDNA) are indicated. wtR, Φ29 DNA right origin of replication (29 bp); wtL, Φ29 DNA left origin of replication (29 bp), GA-1L, GA-1 left origin of replication (29 bp); as in Figure 1, two capital letters indicate the first three nucleotides of the displaced strand and the first three nucleotides of the template strand, respectively; in addition to that, superindexes alone (A/TNNN) denote the sequences from positions 4–6 of the displaced strand if other than wt and paired. Superindexes and subindexes () denote the sequences from positions 4–6 of the displaced and template strands, respectively, when they are unpaired (see scheme above Figure 1B).
First, we checked with appropriate markers that the size of the product DNA linked to the TP was the correct one, this is 29 nucleotides long (see Supplementary Figure S1). We also checked that the level of initiation or replication produced from the non-origin DNA end was negligible (not shown). The results (Figure 1A) showed that the level of full-length oligomer replication is very low for template strands that have purines at the 3′ end (N/A or N/G, being N any nucleotide), intermediate for wild-type templates (N/T) and highest for C-templates (N/C) with the exception of the pairing G/C. Regarding the non-template strand, there are no significative differences for any of the combinations with the wild-type template strand, whether they are paired or not. However, for the CCC-ended templates we observe the highest activity for the combination with AAA at the displaced strand (lane A/C, 18-fold respect to the wild-type), a somewhat lower activity but still 7-fold over the wild-type for origins with TTT or CCC at the displaced strand (lanes T/C and C/C), and a comparatively low activity (0.5 times the wild-type) for the G/C paired origin. This suggests that there is a negative effect for the pairing G/C, and a clear positive effect for the unpairings C/C, T/C and especially A/C. In the case of the A/C template there are two weak initiation bands even when the labeled nucleotide is dATP, not dGTP; we consider these bands as errors of incorporation since the total level of TP-DNA product is particularly high for this template.