Bacteriophage T5 5'-->3' exonuclease is a member of a family of sequence related 5'-nucleases which play an essential role in DNA replication. The 5'-nucleases have both exonucleolytic and structure-specific endo-nucleolytic 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 hairpin substrate. The products of DNA hydrolysis are characterised by mass spectrometry. The steady-state catalytic parameters of the enzyme are reported and it is concluded that T5 5'-->3' exonuclease accelerates the cleavage of a specific phosphodiester bond by a factor of at least 10(15). The catalytic assay has been extended to three mutants of T5 5'-->3' exonuclease, K83A, K196A and K215A. Mutation of any of these three lysine residues to alanine is detrimental to catalytic efficiency. All three lysines contribute to ground state binding of the substrate. In addition, K83 plays a significant role in the chemical reaction catalysed by this enzyme. Possible roles for mutated lysine residues are discussed.
T5 5-3-exonuclease is a member of a family of homologous 5-nucleases essential for DNA replication and repair. We have measured the variation of the steady state parameters of the enzyme with pH. The log of the association constant of the enzyme and substrate is pH-independent between pH 5 and 7, but at higher pH, it decreases (gradient ؊0.91 ؎ 0.1) with increasing pH. The log of the turnover number increases (gradient 0.9 ؎ 0.01) with increasing pH until a pH-independent plateau is reached. The T5 5-3-exonuclease-catalyzed reaction requires the protonation of a single residue for substrate binding, whereas k cat depends on a single deprotonation as demonstrated by the bell-shaped dependence of log (k cat /K m ) on pH. To investigate the role of a conserved lysine (Lys-83), the pH profile of log (k cat /K m ) of a K83A mutant was determined and found to increase with pH (gradient 1.01 ؎ 0.01) until a pH-independent plateau is reached. We therefore conclude that protonation of Lys-83 in the wild type protein facilitates DNA binding. The origin of the pH dependence of the k cat parameter of the wild type enzyme is discussed.Organisms as diverse as bacteriophage and humans require 5Ј-nucleases to participate in, and ensure the fidelity of, DNA replication and repair. These enzymes are involved in excising damaged DNA and the removal of Okazaki fragments produced during replication. T5 5Ј-3Ј-exonuclease is a virus-encoded member of a family of homologous 5Ј-nucleases which includes the N-terminal domain of DNA polymerase I proteins (1). More distantly related eukaryotic and mammalian enzymes also exhibit sequence homology, albeit to a lesser extent, and include the human enzyme hFEN-1 1 (1). The 5Ј-nucleases catalyze the exonucleolytic hydrolysis of phosphodiester bonds in a variety of nucleic acid substrates, including single-stranded and double-stranded DNA and DNA-RNA hybrids (RNase H activity).In addition, the 5Ј-nucleases also exhibit structure-specific endonucleolytic activity, resulting in the cleavage of DNA flap and pseudo-Y structures (Fig. 1a) (2-4). This structure-specific hydrolysis of bifurcated structures is mainly observed at the junction between double-stranded and single-stranded DNA (2-4). Both exonucleolytic and endonucleolytic hydrolyses proceed with scission of the 3Ј-oxygen phosphorus bond to generate products terminating in a 3Ј-hydroxyl group and a 5Ј-phosphate monoester, respectively (5-7).The x-ray crystal structures of five members of the 5Ј-nuclease family have been reported in the absence of DNA substrates (8 -12). The structures of T4 RNase H and the 5Ј-exonuclease domain of Taq polymerase I both contained areas of disorder (10, 12), but the structure of T5 5Ј-3Ј-exonuclease revealed the presence of a hole bounded by a helical arch upon a globular domain containing the putative active site (9). The hole is large enough to accommodate single-but not doublestranded DNA. To explain substrate processing, a threading mechanism in which the single-stranded 5Ј terminus enters the arch has bee...
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