Quantitation of metal ion and DNA junction binding to the Holliday junction endonuclease Cce1.
Cce1 is a magnesium-dependent Holliday junction endonuclease involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae. Cce1 binds four-way DNA junctions as a dimer, opening the junction into an extended, 4-fold symmetric structure, and resolves junctions by the introduction of paired nicks in opposing strands at the point of strand exchange. In the present study, we have examined the interactions of wild-type Cce1 with a noncleavable four-way DNA junction and metal ions (Mg(2+) and Mn(2+)) using isothermal titration calorimetry, EPR, and gel electrophoresis techniques. Mg(2+) or Mn(2+) ions bind to Cce1 in the absence of DNA junctions with a stoichiometry of two metal ions per Cce1 monomer. Cce1 binds to four-way junctions with a stoichiometry of two Cce1 dimers per junction molecule in the presence of EDTA, and one dimer of Cce1 per junction in 15 mM magnesium. The presence of 15 mM Mg(2+) dramatically reduces the affinity of Cce1 for four-way DNA junctions, by about 900-fold. This allows an estimation of DeltaG degrees for stacking of four-way DNA junction 7 of -4.1 kcal/mol, consistent with the estimate of -3.3 to -4.5 kcal/mol calculated from branch migration and NMR experiments [Overmars and Altona (1997) J. Mol. Biol. 273, 519-524; Panyutin et al. (1995) EMBO J. 14, 1819-1826]. The striking effect of magnesium ions on the affinity of Cce1 binding to the four-way junction is predicted to be a general one for proteins that unfold the stacked X-structure of the Holliday junction on binding.
Kvaratskhelia
M
M
Department of Biochemistry, University of Dundee, U.K.
George
S J
SJ
Cooper
A
A
White
M F
MF
D001665:Binding Sites; D002151:Calorimetry; D002413:Cations, Divalent / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000737:chemistry*; D045565:Holliday Junction Resolvases; D008274:Magnesium / Q000737:chemistry*; D008345:Manganese / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship
D002413:Cations, Divalent; D029701:Saccharomyces cerevisiae Proteins; D008345:Manganese; D004247:DNA; D004706:Endodeoxyribonucleases; C473486:CCE1 protein, S cerevisiae; D045565:Holliday Junction Resolvases; D008274:Magnesium