The influence of reducing agent and 1,10-phenanthroline concentration on DNA cleavage by phenanthroline + copper

Abstract

Copper in the presence of excess 1,10-phenanthroline, a reducing agent, and molecular oxygen causes cleavage of DNA with a preference for T-3',5'-A-steps, particularly in TAT triplets. The active molecular species is commonly thought to be the bis-(1,10-phenanthroline)Cu(I) complex, (Phen)2Cu(I), regardless of the reducing agent type. We have found that (Phen)2Cu(I) is not the predominant copper complex when 3-mercaptopropionic acid (MPA) or 2-mercaptoethanol are used as the reducing agents, but (Phen)2Cu(I) predominates when ascorbate is used as the reducing agent. Substitution of ascorbate for thiol significantly enhances the rate of DNA cleavage by 1,10-phenanthroline + copper, without altering the sequence selectivity. We show that (Phen)2Cu(I) is the complex responsible for DNA cleavage, regardless of reducing agent, and that 1,10-phenanthroline and MPA compete for copper coordination sites. DNA cleavage in the presence of ascorbate also occurs under conditions where the mono-(1,10-phenanthroline)Cu(I) complex predominates (1:1 phenanthroline:copper ratio), but preferential cleavage was observed at a CCGG sequence and not at TAT sequences. The second phenanthroline ring of the (Phen)2Cu(I) complex appears essential for determining the T-3',5'-A sequence preferences of phenanthroline + copper when phenanthroline is in excess.