Date Published: May 20, 2019
Publisher: Public Library of Science
Author(s): Iván de la Torre, Victor Quiñones, Margarita Salas, Alicia del Prado, Domenico Maiorano.
The genome of Bacillus subtilis phage ϕ29 consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5’ end (TP-DNA). ϕ29 DNA polymerase is the enzyme responsible for viral DNA replication, due to its distinctive properties: high processivity and strand displacement capacity, being able to replicate the entire genome without requiring the assistance of processivity or unwinding factors, unlike most replicases. ϕ29 single-stranded DNA binding protein (SSB) is encoded by the viral gene 5 and binds the ssDNA generated in the replication of the ϕ29 TP-DNA. It has been described to stimulate the DNA elongation rate during the DNA replication. Previous studies proposed residues Tyr50, Tyr57 and Tyr76 as ligands of ssDNA. The role of two of these residues has been determined in this work by site-directed mutagenesis. Our results showed that mutant derivative Y57A was unable to bind to ssDNA, to stimulate the DNA elongation and to displace oligonucleotides annealed to M13 ssDNA, whereas mutant Y50A behaved like the wild-type SSB.
Bacteriophage ϕ29 is a lytic phage that infects Bacillus subtilis. Its genome consists of a linear double stranded DNA (dsDNA) molecule of 19,285 base pairs (bp) with a terminal protein (TP) covalently linked to each 5’ DNA end (parental TP). The minimal replication origin is formed by the parental TP together with the terminal 12 bp containing a 6 bp inverted terminal repeat (3’-TTTCAT) . The ϕ29 DNA polymerase forms a heterodimer with a free TP molecule (primer TP) that recognizes the replication origins. The ϕ29 double-stranded DNA binding protein (DBP) binds all along ϕ29 DNA, forming a nucleoprotein complex at the replication origins that has been proposed to open the DNA ends, facilitating the initiation step of replication . Once the replication origin is recognized by the heterodimer, the ϕ29 DNA polymerase catalyses the formation of a phosphoester bond between the initiating dAMP and the hydroxyl group of Ser232 of the TP . The initiation occurs opposite the 3’ second nucleotide of the template strand. This TP-dAMP complex is translocated backwards one position to recover the template information corresponding to the first 3’-T . There is a transition stage between the initiation with TP and the elongation with the DNA. When the polymerase reaches the tenth nucleotide, it is dissociated from the TP  and the same polymerase catalyses processive chain elongation of the complete ϕ29 genome via a strand displacement mechanism .