Terminal protein-primed DNA amplification.

Abstract

By (6, 7). In this reaction, dATP is selected by base complementarity with the second 3'-nucleotide of the template strand (8). After this initiation step, dissociation of the TP-DNA polymerase heterodimer is likely to occur (transition) to replace the TP-DNA polymerase interactions required for initiation by the DNA polymerase-DNA interactions required for the elongation of the newly created DNA primer. Concomitantly, an asymmetric translocation (sliding back) of only TP-dAMP, but not of the template, followed by addition of a new dAMP residue, allows the recovery of the information corresponding to the first template nucleotide (8). During elongation, 429 DNA polymerase catalyzes highly processive polymerization coupled to strand displacement (9), and, therefore, complete replication of both strands proceeds continuously from each terminal priming event. As the two replication forks move, DNA synthesis is initially coupled to strand displacement of long stretches of single-stranded 429 DNA, producing type I replicative intermediates (see Fig. 1 (10,11). Protein p6 (Mr = 11,873) and protein p5 (Mr = 13,212), obtained from 429-infected B. subtilis cells, were purified as described (12, 13). TP-linked DNA from 429 susl4 (1242)

DNA polymerase template switching at specific sites on the φ29 genome causes the in vivo accumulation of subgenomic φ29 DNA molecules

“…An in vitro terminal protein (TP)-primed ᮊ 1998 Blackwell Science Ltd, Molecular Microbiology, 29, 787-798 e. The genomes of BS32 and 15 are only partly sequenced and therefore they may contain more of these DSEs. require, in addition to the TP-DNA, the following four 29-encoded proteins: DNA polymerase, TP, SSB and DBP (Blanco et al, 1994). In the absence of SSB, however, subgenomic 29 DNA molecules were generated (Esteban et al, 1997).…”

“…An efficient in vitro 29 DNA amplification system has been established that requires, besides purified 29 DNA polymerase and 29 template DNA, the phage-encoded terminal protein (TP), single-stranded DNA-binding protein (SSB) and double-stranded DNA-binding protein (DBP) (Blanco et al, 1994). However, under suboptimal in vitro replication conditions, i.e.…”

Phage φ29 protein p56 prevents viral DNA replication impairment caused by uracil excision activity of uracil-DNA glycosylase

“…This amplification system (15) requires, in addition to TP and DNA polymerase, 29 ssDNA binding protein (SSB) (to preclude the appearance of short palindromic DNAs) and 29 dsDNA binding protein (DBP) (for optimal activation of the origins). Thus, in the presence of these four proteins, numerous consecutive replication rounds occur, mimicking the natural amplification of viral DNA.…”

Terminal protein-primed amplification of heterologous DNA with a minimal replication system based on phage Φ29

“…The Φ29 DNA polymerase is endowed with a potent strand displacement activity and a high processivity that allows it to perform the 19-kb replication of the phage genome from a single binding event and without the need for any accessory factors (12). The Φ29 replication system has been reported to amplify the genome of the phage (TP-DNA) to microgram amounts starting from nanograms in a single-step isothermal reaction (10). It is also important to note that the amplification of the 19-kb phage DNA takes place with high fidelity yielding fully functional DNA (10).…”

“…The Φ29 in vitro minimal amplification system of TP-DNA is based on four essential components, DNA polymerase, TP, double-stranded DNA binding protein p6, and single-stranded DNA binding protein (SSB) p5 (10). In the Φ29 DNA replication mechanism (13) (Fig.…”