The enzymatic basis of processivity in lambda exonuclease

λ Exonuclease is a highly processive 5′→3′ exonuclease that degrades double-stranded (ds)DNA. The single-stranded DNA produced by λ exonuclease is utilized by homologous pairing proteins to carry out homologous recombination. The extensive studies of λ biology, λ exonuclease enzymology and the availability of the X-ray crystallographic structure of λ exonuclease make it a suitable model to dissect the mechanisms of processivity. λ 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 λ exonuclease involved in recognizing the 5′-phosphate at the ends of broken dsDNA. The preference of λ 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 λ exonuclease mutant impaired in 5′-phosphate recognition, the significance of catalytic efficiency in modulating the processivity of λ exonuclease has been elucidated. We propose a model in which processivity of λ exonuclease is expressed as the net result of competition between pathways that either induce forward translocation or promote reverse translocation and dissociation.