RECOMBINATION BETWEEN COLIPHAGES LAMBDA AND PHI-80.

The integrase protein (Int) of phage lambda is a well-studied representative of the tyrosine recombinase family, whose defining features are two sequential pairs of DNA cleavage/ligation reactions that proceed via a 3' phosphotyrosine covalent intermediate to first form and then resolve a Holliday junction recombination intermediate. We devised an assay that takes advantage of DNA hairpin formation at one Int target site to trap Int cleavages at a different target site, and thereby reveal iterative cycles of cleavage and ligation that would otherwise be undetected. Using this assay and others to compare wild-type Int and a mutant (R169D) defective in forming proper dimer/tetramer interfaces, we found that the efficiency of "bottom-strand" DNA cleavage by wild-type Int, but not R169D, is very sensitive to the base-pair at the "top-strand" cleavage site, seven base-pairs away. We show that this is related to the finding that hairpin formation involving ligation of a mispaired base is much faster for R169D than for wild-type Int, but only in the context of a multimeric complex. During resolution of Holliday junction recombination intermediates, wild-type Int, but not R169D, is very sensitive to homology at the sites of ligation. A long-sought insight from these results is that during Holliday junction resolution the tetrameric Int complex remains intact until after ligation of the product helices has been completed. This contrasts with models in which the second pair of DNA cleavages is a trigger for dissolution of the recombination complex.

Bacteriophage lambda↗

PubMed2004

Quantitative analysis of a RNA-cleaving DNA catalyst obtained via in vitro selection.

In vitro selections performed in the presence of Mg(2+) generated DNA sequences capable of cleaving an internal ribonucleoside linkage. Several of these, surprisingly, displayed intermolecular catalysis and catalysis independent of Mg(2+), features that the selection protocol was not explicitly designed to select. A detailed physical organic analysis was applied to one of these DNAzymes, termed 614. First, the progress curve for the reaction was dissected to identify factors that prevented the molecule from displaying clean first-order transformation kinetics and 100% conversion. Several factors were identified and quantitated, including (a) competitive intra- and intermolecular rate processes, (b) alternative reactive and unreactive conformations, and (c) mutations within the catalyst. Other factors were excluded, including "approach to equilibrium" kinetics and product inhibition. The possibility of complementary strand inhibition was demonstrated but was shown to not be a factor under the conditions of these experiments. The rates of the intra- and intermolecular processes were compared, and saturation models for the intermolecular process were built. The rate-limiting step for the intermolecular reaction was found to be the association/folding of the enzyme with the substrate and not the cleavage step. The DNAzyme 614 is more active in trans than in cis and more active at temperatures below the selection temperature than at the selection temperature. Many of these properties have not been reported in similar systems; these results therefore expand the phenomenology known for this class of DNA-based catalysts. A brief survey of other catalysts arising from this selection found other Mg(2+)-independent DNAzymes and provided a preliminary view of the ruggedness of the landscape, relating function to structure in sequence space. Hypotheses are suggested to account for the fact that a selection in the presence of Mg(2+) did not exploit this Mg(2+). This study of a specific catalytically active DNAzyme is an example of studies that will be necessary generally to permit in vitro selection to help us understand the distribution of function in sequence space.

Bacteriophage lambda↗