Structural requirements of substrate DNA for binding to and cleavage by RuvC, a Holliday junction resolvase.
To elucidate the molecular mechanism of the resolution of Holliday junctions by Escherichia coli RuvC protein, we studied biochemical properties of the protein using various synthetic DNA junctions as model substrates. RuvC cleaves not only a four-way junction but also three-way junctions efficiently. The central core of homology in the junction is essential for the substrates to be cleavable by RuvC. Although the divalent cations are essential for the endonuclease activity, RuvC efficiently forms specific complexes with four-way junctions in the absence of the cations, irrespective of the presence of homologous core sequences. By using T7 endonuclease I as a probe, we studied the topology of the substrate junctions used in our study. The results suggest that RuvC cleaves the three-way junctions with homology core when they become four-way conformers. From the present studies, we propose that RuvC initially binds mostly nonproductively to four-way junctions, which does not require divalent metals, and subsequently cleaves the junctions by a mechanism dependent on a divalent cation and a particular topological conformer that is induced by the sequences at the mobile junctions.
Takahagi
M
M
Department of Experimental Chemotherapy, Osaka University, Japan.
Iwasaki
H
H
Shinagawa
H
H
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004706:Endodeoxyribonucleases; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009713:Nucleotidyltransferases / Q000378:metabolism*; D019895:Transposases
D001426:Bacterial Proteins; D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C069806:ruvC protein, E coli; D004247:DNA; D009713:Nucleotidyltransferases; D019895:Transposases; D004706:Endodeoxyribonucleases