Published online 12 October 2004
Nucleic Acids Research, Vol. 32 No. 18 © Oxford University Press 2004; all rights reserved
Substrate recognition and catalysis by the Holliday junction resolving enzyme Hje
Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK and 1 Centre for Biomolecular Sciences, University of St Andrews, North Haugh, St Andrews, Fife KY16 9ST, UK
* To whom correspondence should be addressed: Tel: +44 1382 348325; Fax: +44 1382 345764; Email: C.S.Bond{at}dundee.ac.uk
Correspondence may also be addressed to Malcolm White. Tel: +44 1334 463432; Fax: +44 1334 462595; Email: mfw2{at}st-and.ac.uk
Present address: Derek J. Richard, Queensland Institute of Medical Research, PO Royal Brisbane Hospital, Queensland 4029, Australia
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received August 31, 2004; Revised and Accepted September 16, 2004
Two archaeal Holliday junction resolving enzymes, Holliday junction cleavage (Hjc) and Holliday junction endonuclease (Hje), have been characterized. Both are members of a nuclease superfamily that includes the type II restriction enzymes, although their DNA cleaving activity is highly specific for four-way junction structure and not nucleic acid sequence. Despite 28% sequence identity, Hje and Hjc cleave junctions with distinct cutting patterns—they cut different strands of a four-way junction, at different distances from the junction centre. We report the high-resolution crystal structure of Hje from Sulfolobus solfataricus. The structure provides a basis to explain the differences in substrate specificity of Hje and Hjc, which result from changes in dimer organization, and suggests a viral origin for the Hje gene. Structural and biochemical data support the modelling of an Hje:DNA junction complex, highlighting a flexible loop that interacts intimately with the junction centre. A highly conserved serine residue on this loop is shown to be essential for the enzyme's activity, suggesting a novel variation of the nuclease active site. The loop may act as a conformational switch, ensuring that the active site is completed only on binding a four-way junction, thus explaining the exquisite specificity of these enzymes.
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