Quantitation of metal ion and DNA junction binding to the Holliday junction endonuclease Cce1

Cce1 is a magnesium-dependent Holliday junction endonuclease involved in th e 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 introdu ction 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 w ith a noncleavable four-way DNA junction and metal ions (Mg2+ and Mn2+) usi ng isothermal titration calorimetry, EPR, and gel electrophoresis technique s. Mg2+ or Mn2+ ions bind to Cce1 in the absence of DNA junctions with a st oichiometry of two metal ions per Cce1 monomer. Cce1 binds to four-way junc tions 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. Th e presence of 15 mM Mg2+ dramatically reduces the affinity of Cce1 for four -way DNA junctions, by about 900-fold. This allows an estimation of Delta G degrees for stacking of four-way DNA junction 7 of -4.1 kcal/mol, consiste nt with the estimate of -3.3 to -4.5 kcal/mol calculated from branch migrat ion 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-struct ure of the Holliday junction on binding.