The
flap endonucleases, or 5' nucleases, are involved in DNA replication and repair. They possess both 5'-3' exonucleolytic activity and the ability to cleave bifurcated, or branched
DNA, in an endonucleolytic, structure-specific manner. These
enzymes share a great degree of structural and sequence similarity. Conserved
acidic amino acids, whose primary role appears to be chelation of essential divalent
cation cofactors, lie at the base of the active site. A loop, or helical archway, is located above the active site. A conserved
tyrosine residue lies at the base of the archway in phage
T5 flap endonuclease. This residue is conserved in the structures of all
flap endonucleases analysed to date. We mutated the
tyrosine 82
codon in the cloned T5 5' nuclease to one encoding
phenylalanine. Detailed analysis of the purified Y82F
protein revealed only a modest (3.5-fold) decrease in binding affinity for
DNA compared with wild-type in the absence of cofactor. The modified nuclease retains both structure-specific
endonuclease and
exonuclease activities. Kinetic analysis was performed using a newly developed single-cleavage assay based on hydrolysis of a fluorescently labelled
oligonucleotide substrate. Substrate and products were resolved by denaturing HPLC. Steady-state kinetic analysis revealed that loss of the
tyrosine hydroxyl function did not significantly impair k(cat). Pre-steady state analysis under single-turnover conditions also demonstrated little change in the rate of reaction compared to the wild-type
protein. The pH dependence of the kinetic parameters for the Y82F
enzyme-catalysed reaction was bell-shaped as for the wild-type
protein. Thus, Y82 does not play a role in catalysis. However, steady-state analysis did detect a large (approximately 300-fold) defect in K(M). These results imply that this conserved
tyrosine plays a key role in ternary complex formation (
protein-
DNA-
metal ion), a prerequisite for catalysis.