Protein-Primed Replication of Bacteriophage phi29 DNA

Unlike circular genomes in which the conventional replication fork machinery can accomplish the copy of the complete molecule, the requirement of a DNA/RNA molecule to prime DNA synthesis imposes replication strategies to avoid the loss of genetic information contained at the very end of the lagging strand in linear chromosomes since DNA polymerases are unable to start de novo DNA synthesis. Thus, once the most terminal primer is removed, a short region of unreplicated single-stranded DNA (ssDNA) will remain at the end of the chromosome that would eventually lead to a continuous shortening of the daughter DNA molecule after successive rounds of DNA replication (the end-replication problem). Therefore, it is essential to guarantee replication of the chromosome ends, that otherwise would cause cell death. Organisms containing linear genomes have developed novel replication strategies to overcome such a problem by either yielding head–tail concatemers, most of them making use of terminal redundancies as phages T4, T7 and SPP1, or by the circularisation and further rolling circle replication of their chromosomes, as it occurs in phage [reviewed in (Salas & de Vega, 2008)]. In higher eukaryotes telomerase extends directly the 3´ end, producing an overhanged ssDNA end (Kornberg & Baker, 1992) that finally can invade homologous double-stranded telomeric tracts, enlarging and protecting chromosome ends (Verdun & Karlseder, 2007). Other organisms, as bacteriophages, animal viruses as adenovirus and human hepatitis B virus, mitochondrial plasmids, linear chromosomes and plasmids of Streptomyces (Salas, 1999), as well as several virus infecting Archaea, as halovirus (Bamford et al., 2005; Bath et al., 2006), possess replication origins, constituted by inverted terminal repetitions (ITR) together with a terminal protein (TP), placed at both ends of their linear chromosomes (Salas, 1991). In these cases, the location of the two replication origins allows both strands to be replicated continuously, without requiring asymmetric complexes of the replicative DNA polymerase with other accessory proteins to control the different mechanics of continuous and discontinuous synthesis (Blanco et al., 1989). The TP provides the OH- group of a specific serine, threonine or tyrosine to prime initiation of DNA replication from the ends of the linear chromosome, circumventing the end replication problem, the TP remaining covalently linked to such 5´-termini of the genome (TP-DNA) (Salas, 1991, 1999; Salas et al.,1996).Peer reviewe