Differential binding of Mn(II) to bacteriophage T5 5′ nuclease and vital role of metal site II in exonucleolytic cleavage

Abstract

5'-3' exonucleases are essential for D N A replication, recombination and repair. T5 5' nuclease is a member of this group which exhibit 5'-3' exonuclease and structure-specific endonuclease activity. Our previous studies suggested that endo-and exonuclease activity are separable (1,2). Site-directed mutagenesis and biochemical analyses were used to investigate the role of absolutely conserved residues situated near the metal-binding sites in T5 5' nuclease. For example, mutations of a conserved tyrosine were found to increase the k, 4-45 fold (wild-type has k, of 10 nM) but retained full endo-and 5-15% exo-nuclease activity. Variation in the steady state kinetic parameters of wildtype and mutant T5 5' nuclease with p H demonstrated that protonation of Lys-83 is critical for DNA-binding (3). The results suggest that the endo-and exo-nucleolytic activities of the T5 enzyme are distinct and utilise different mechanisms to effect substrate cleavage.The 5' nucleases are required for both DNA replication and repair. T5 5' nuclease is a member of this group which exhibit 5'-3' exonuclease and structure-specific endonuclease activity. It is proposed that single-stranded DNA threads through a hole observed in the X-ray structure of such nucleases '.Structure-function studies suggest that different mechanisms for endo-and exonucleolytic hydrolysis are used by this enzyme*. Site-directed mutagenesis was employed to investigate the role of two acidic and two basic residues thought to be involved in cofactor and substrate binding respectively. These conserved residues form an integral part of a helix-loop-helix feature ', representing residues 189-224 of the T5 exonuclease sequence. Using quantitative band shift assays with different substrates and mutant proteins we determined the impact of mutation upon binding and catalysis. Our data lead us to propose an alternative DNA binding model for T5 exonuclease.Nucleic Acids Res., 25:4224-4229 382~90-93 29 Differential binding of Mn(I1) to bacteriophage T5 5' nuclease and vital role of metal site I1 in exonucleolytic cleavage 5' nucleases are essential for DNA replication and repair. They catalyze exonucleolytic hydrolysis of phosphodiester bonds in a variety of nucleic acid, also exhibit structure-specific endonucleolytic activity, allowing them to cleave bifucations of flap structures containing free 5'-end. Crystallographic and biochemical studies suggest single-stranded substrates become threaded through 5' nucleases, where hydrolysis is affected with the aid of divalent metal cations. Here we show the T5 5' nuclease to be promiscuous in metal ions as cofactor. By varying concentrations of various transition metals the cleavage pattern can be dramatically changed so that less than 1% of the products were caused by exonucleolytic cleavage. ITC data demenstrate differential binding of Mn(I1) to wild-type nuclease with total stochiometry of 2, while Co(I1) can occupy site I with stochiometry of 1 only. Mutagenesis of two aspatate prevents Mn(I1) from occupying metal site 11, thi...