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Nucleic Acids Research, 2001, Vol. 29, No. 13 2772-2779
© 2001 Oxford University Press

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

Scott J. Garforth, Dipak Patel, Min Feng and Jon R. Sayers* Division of Genomic Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield S10 2RX, UK

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.

* To whom correspondence should be addressed. Tel: +44 114 271 2327; Fax: +44 114 273 9926; Email: j.r.sayers{at}sheffield.ac.ukPresent address:Scott J. Garforth, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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