Tyrosines involved in the activity of φ29 singlestranded DNA binding protein

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.