Parallel symmetric immobile DNA junctions as substrates for E. coli RuvC Holliday junction resolvase.

Abstract

RuvC is a well-characterized Holliday junction resolvase from E. coli. The presence of symmetry in its preferred recognition sequence leads to ambiguity in the position of the crossover point in the junction, because a symmetric junction can undergo branch migration. Symmetric immobile junctions are junctions that contain such symmetric sites, but are prevented from migrating by their physical characteristics. RuvC activity had been analyzed previously by traditional symmetric immobile junctions, in which the helix axes are held antiparallel in a double-crossover motif. Bowtie junctions are branched four-arm molecules containing 5',5' and 3',3' linkages at their crossover points. A new type of symmetric immobile junction can be made by flanking the crossover point of a Bowtie junction with a symmetric sequence. The junction is immobile because mobility would lead to pairing between parallel, rather than antiparallel, nucleotide pairs. In contrast to conventional Holliday junctions and their analogues, the Bowtie junction assumes a parallel, rather than antiparallel, helical domain conformation, offering a new type of substrate for Holliday junction resolvases. Here, we report the digestion of Bowtie junctions by RuvC. We demonstrate that Bowtie junctions can function as symmetric immobile junctions in this system. We also show that RuvC cleaves antiparallel junctions much more efficiently than parallel junctions, where the protein can bind (and cleave) only one site at a time. These data suggest that the presence of two binding sites leads to communication between the two subunits of the enzyme to increase its activity.