Holliday　junction　resolution　is　a　crucial　process　in　homologous　recombination　and　DNA　double-strand　break　repair.　Complete　Holliday　junction　resolution　requires　two　stepwise　incisions　across　the　center　of　the　junction,　but　the　precise　mechanism　of　metal　ion-catalyzed　Holliday　junction　cleavage　remains　elusive.　Here,　we　perform　a　metal　ion-triggered　catalysis　in　crystals　to　investigate　the　mechanism　of　Holliday　junction　cleavage　by　MOC1.　We　capture　the　structures　of　MOC1　in　complex　with　a　nicked　Holliday　junction　at　various　catalytic　states,　including　the　ground　state,　the　one-metal　ion　binding　state,　and　the　two-metal　ion　binding　state.　Moreover,　we　also　identify　a　third　metal　ion　that　may　aid　in　the　nucleophilic　attack　on　the　scissile　phosphate.　Further　structural　and　biochemical　analyses　reveal　a　metal　ion-mediated　allosteric　regulation　between　the　two　active　sites,　contributing　to　the　enhancement　of　the　second　strand　cleavage　following　the　first　strand　cleavage,　as　well　as　the　precise　symmetric　cleavage　across　the　Holliday　junction.　Our　work　provides　insights　into　the　mechanism　of　metal　ion-catalyzed　Holliday　junction　resolution　by　MOC1,　with　implications　for　understanding　how　cells　preserve　genome　integrity　during　the　Holliday　junction　resolution　phase.　©　The　Author(s)　2024.