The enzymatic basis of processivity in λ exonuclease

Abstract

λ Exonuclease is a highly processive 5��3� exonuclease that degrades double-stranded (ds)DNA. The single-stranded DNA produced by exonuclease is utilized by homologous pairing proteins to carry out homologous recombination. The extensive studies of biology, exonuclease enzymology and the availability of the X-ray crystallographic structure of exonuclease make it a suitable model to dissect the mechanisms of processivity. Exonuclease is a toroidal homotrimeric molecule and this quaternary structure is a recurring theme in proteins engaged in processive reactions in nucleic acid metabolism. We have identified residues in exonuclease involved in recognizing the 5�-phosphate at the ends of broken dsDNA. The preference of exonuclease for a phosphate moiety at 5� dsDNA ends has been established in previous studies; our results indicate that the low activity in the absence of the 5�-phosphate is due to the formation of inert enzyme-substrate complexes. By examining a exonuclease mutant impaired in 5�-phosphate recognition, the significance of catalytic efficiency in modulating the processivity of exonuclease has been elucidated. We propose a model in which processivity of exonuclease is expressed as the net result of competition between pathways that either induce forward translocation or promote reverse translocation and dissociation. © Oxford University Press 2001.