Mechanistic studies of a 5′ nuclease from T5 bacteriophage

Bacteriophage T5 5' nuclease is a member of a family of sequence related 5' nucleases which play essential roles in DNA replication. The 5' nucleases have both exonucleolytic and structure-specific endonucleolytic DNA cleavage activity and are conserved in organisms as diverse as bacteriophage and mammals. Here, we report the development of a structure-specific single-cleavage assay for this enzyme which uses a 5'-overhanging DNA hairpin substrate. The assay has been used to characterise mutant enzymes and to determine the pH dependence of the kinetic parameters of the enzyme allowing insights into the mechanism of this class of enzymes.