Abstract
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.
Highlights
Bacteriophage φ29 is a lytic phage that infects Bacillus subtilis
The φ29 DNA polymerase forms a heterodimer with a free terminal protein (TP) molecule that recognizes the replication origins
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 [3]
Summary
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 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 [2]. 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 [3]. 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 [4].
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
