STRUCTURAL RECOGNITION AND DISTORTION BY THE DNA JUNCTION-RESOLVING ENZYME RUSA

RusA is a relatively small DNA junction-resolving enzyme of lambdoid p hage-origin. Many of the physical characteristics of this enzyme are s imilar to those of junction-resolving enzymes of different origins. Ru sA binds to DNA junctions as a dimer, with a dissociation constant of 2 to 7 nM. RusA also exists in dimeric form in free solution, with a h alf time for subunit exchange of 4.2 minutes. We find that RusA can cl eave both fixed junctions and those that can undergo a number of steps of branch migration, and confirm that the enzyme exhibits a strong pr eference for cleavage 5' to a CpC sequence. We have isolated a mutant protein, RusA D70N, that is completely inactive in cleavage while bind ing normally to DNA junctions, suggesting a role for aspartate 70 in t he cleavage reaction. Constraining the conformation of the junction by means of tethering the helical ends leads to a marked reduction in cl eavage rate by RusA, suggesting that the structure must be altered for cleavage. Using comparative gel electrophoresis we find that the glob al structure of the DNA junction is altered on RusA binding, into a st ructure that is different from any that is formed by the free junction . Moreover, the structure of the complex is the same irrespective of t he presence or absence of magnesium ions. Thus, like all the junction- resolving enzymes, RusA both recognises and distorts the structure of DNA junctions. (C) 1998 Academic Press Limited.