Structural recognition and distortion by the DNA junction-resolving enzyme RusA.
RusA is a relatively small DNA junction-resolving enzyme of lambdoid phage-origin. Many of the physical characteristics of this enzyme are similar to those of junction-resolving enzymes of different origins. RusA 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 half time for subunit exchange of 4.2 minutes. We find that RusA can cleave both fixed junctions and those that can undergo a number of steps of branch migration, and confirm that the enzyme exhibits a strong preference for cleavage 5' to a CpC sequence. We have isolated a mutant protein, RusA D70N, that is completely inactive in cleavage while binding normally to DNA junctions, suggesting a role for aspartate 70 in the cleavage reaction. Constraining the conformation of the junction by means of tethering the helical ends leads to a marked reduction in cleavage rate by RusA, suggesting that the structure must be altered for cleavage. Using comparative gel electrophoresis we find that the global structure of the DNA junction is altered on RusA binding, into a structure that is different from any that is formed by the free junction. Moreover, the structure of the complex is the same irrespective of the presence or absence of magnesium ions. Thus, like all the junction-resolving enzymes, RusA both recognises and distorts the structure of DNA junctions.
Giraud-Panis
M J
MJ
Department of Biochemistry, The University of Dundee, Dundee, DD1 4HN, UK.
Lilley
D M
DM
D002384:Catalysis; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D045565:Holliday Junction Resolvases; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D004706:Endodeoxyribonucleases; D045565:Holliday Junction Resolvases; C112057:RusA protein, E coli