Unusually wide co-factor tolerance in a metalloenzyme; divalent metal ions modulate endo-exonuclease activity in T5 exonuclease

T5 5 ' -3 ' exonuclease is a member of a homologous group of 5 ' nucleases which require divalent metal co-factors, Structural and biochemical studies suggest that single-stranded DNA substrates thread through a helical arch or hole in the protein, thus bringing the phosphodiester backbone into close proximity with the active site metal co-factors. In addition to the expected use of Mg2+, Mn2+ and Co2+ as co-factors, we found that divalent zinc, iron, nickel and copper ions also supported catalysis, Such a range of co-factor utilisation is unusual in a single enzyme, Some co-factors such as Mn2+ stimulated the cleavage of double-stranded closed-circular plasmid DNA, Such endonucleolytic cleavage of circular double-stranded DNA cannot be readily explained by the threading model proposed for the cleavage of substrates with free 5 ' -ends as the hole observed in the crystal structure of T5 exonuclease is too small to permit the passage of double-stranded DNA, We suggest that such a substrate may gain access to the active site of the enzyme by a process which does not involve threading.