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Nucleic Acids Research 2004 32(21):6176-6186; doi:10.1093/nar/gkh960
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Published online 29 November 2004

Nucleic Acids Research, Vol. 32 No. 21 © Oxford University Press 2004; all rights reserved

A novel nuclease-ATPase (Nar71) from archaea is part of a proposed thermophilic DNA repair system

Colin P. Guy, Alan I. Majerník1, James P. J. Chong1 and Edward L. Bolt*

Institute of Genetics, School of Biology, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK and 1 Department of Biology, University of York, York YO10 5YW, UK

* To whom correspondence should be addressed. Tel: +44 115 9709404; Fax: +44 115 9709906; Email: ed.bolt{at}nottingham.ac.uk
Present address: Alan I. Majerník, Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, 90028 Ivanka pri Dunaji, Slovakia

Received September 29, 2004; Revised and Accepted November 8, 2004

We have identified a novel structure-specific nuclease in highly fractionated extracts of the thermophilic archaeon Methanothermobacter thermautotrophicus (Mth). The 71 kDa protein product of open reading frame mth1090 is a nuclease with ATPase activity, which we call Nar71 (Nuclease-ATPase in Repair, 71 kDa). The nar71 gene is located in a gene neighbourhood proposed by genomics to encode a novel DNA repair system conserved in thermophiles. The biochemical characterization of Nar71 presented here is the first analysis from within this neighbourhood, and it supports the insight from genomics. Nuclease activity of Nar71 is specific for 3' flaps and flayed duplexes, targeting single-stranded DNA (ssDNA) regions. This activity requires Mg2+ or Mn2+ and is greatly reduced in ATP. In ATP, Nar71 displaces ssDNA, also with high specificity for 3' flap and flayed duplex DNA. Strand displacement is weak compared with nuclease activity, but in ATP{gamma}S it is abolished, suggesting that Nar71 couples ATP hydrolysis to DNA strand separation. ATPase assays confirmed that Nar71 is stimulated by ssDNA, though not double-stranded DNA. Mutation of Lys-117 in Nar71 abolished ATPase and nuclease activity, and we describe a separation-of-function mutant (K68A) that has lost ATPase activity but retains nuclease activity. A model of possible Nar71 function in DNA repair is presented.


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