The structural and enzymatic basis of processivity in lambda exonuclease

The purpose of this study was to dissect the mechanism of processive DNA digestion by λ exo as a means to gain insights into how the processivity may be regulated during homologous recombination. The mechanistic basis of processivity was addressed both from a structural and an enzymatic point of view.; λ exo is a homo-trimeric ring-shaped protein with a funnel-shaped channel (It's a Doughnut!). The processivity of λ exo is proposed to result from topological linkage between the λ exo toroid and the ssDNA product of dsDNA digestion (similar to a bead on a string). The topologial model makes the following specific predictions: (1) λ exo should act processively on dsDNA, but distributively on ssDNA, since partially digested dsDNA, but not ssDNA, has the undigested 3′-ending strand that is proposed to thread through the enzyme. (2) Mutants of λ exo that are unable to form trimers, and hence unable to encricle the DNA, but retain catalytic activity, should be distributive. Our results taken together with previous work are consistent with these predictions. The activity of λ exo on ssDNA is indeed distributive. Mutations predicted to destabilize the assembly of λ exo into a ring confer a null phenotype in vivo . The in vitro consequences of these mutations are being tested and preliminary results indicate that the mutations impair both trimerization and processive DNA resection.; To understand the enzymatic basis of the processive nuclease activity of λ exo, the active site of the enzyme and the interaction of the enzyme with dsDNA substrates with varying ends was characterized. The λ exo protein sequence was used to generate a superfamily of conserved viral nucleases. Conserved motifs in the superfamily were hypothesized to identify residues that confer activity and specificity to dsDNA resection. Two conserved residues, D119 and R28, were mutated to test this hypothesis. The behaviour of the mutants in vivo (R28A and D119A) and in vitro (R28A) is consistent with the hypothesis. Moreover, the activities of the wild-type and R28A enzymes on dsDNA with and without a 5′ -phosphate lead us to conclude that R28 belongs to a conserved motif in the λ exo family of nucleases that is involved in recognizing the 5′-phosphate at the ends of broken dsDNA. (Abstract shortened by UMI.)...