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Book of Abstracts: Albany 2003

category image Albany 2003
Conversation 13
Abstract Book
June 17-21 2003

DNA Binding by the BRCT Domain of NAD+-dependent DNA Ligases

DNA ligases catalyse joining of DNA ends, a critical step in the replication, repair and recombination of DNA. As the first step in ligation, all DNA ligases form a covalent enzyme-adenylate intermediate, using either NAD+ or ATP as the donor of the adenylate group (1). The uniqueness of NAD+-dependent DNA ligases to eubacteria makes them an attractive target for novel antibiotics.

High-resolution structures have been obtained for full length NAD+-dependent DNA ligases (LigA) from Thermus filiformis (2) and for the N-terminal adenylation domain of LigA from Bacillus stearothermophilus (3). Within the four distinct domains of LigA are several well-characterised protein folds, including a zinc-finger (Zn), an oligomer-binding (OB) β-barrel, a helix-hairpin-helix motif (HhH) and a BRCT motif. A model for the end-joining action of LigA suggests two regions of the enzyme interact independently with DNA (2). To obtain experimental evidence for the molecular details of DNA binding to this enzyme, we have initiated structural analysis of LigA from Escherichia coli.

The C-terminus of all essential NAD+-dependent DNA ligases contains a BRCT domain, which mediates macromolecular interactions in other proteins. Using in vitro gel-shift assays, we show that the BRCT domain from E. coli LigA promotes formation of a stable complex between LigA and DNA. The BRCT domain is not essential for DNA end-joining activity in vitro or in vivo, though it does improve the efficiency of end-joining in vitro.

This study confirms two aspects of the predictions from the X-ray crystallographic studies of T. filiformis LigA. Firstly, we show that the BRCT domain represents the ?non-catalytic? DNA binding activity of the enzyme (or at least a critical part of it). Secondly, since deletion of the BRCT domain does not completely abolish ligation activity, other parts of LigA must be able to bind to DNA. This latter point confirms that multiple independent DNA binding activities must reside within the protein. Biophysical chemistry techniques are being used to elucidate the modes of interaction between DNA and LigA.

Acknowledgments

The research was supported by grants from the BBSRC (UK) and the University of East Anglia.

Manuel Lavesa-Curto1
Adam Wilkinson2
Andrew Hemmings3
Richard Bowater1

1School of Biological Sciences
University of East Anglia
Norwich NR4 7TJ
United Kingdom
2Phico Therapeutics Ltd
Babraham Hall
Babraham, Cambridge
CB2 4AT, UK
3Schools of Biological Sciences and Chemical Sciences & Pharmacy
University of East Anglia
Norwich NR4 7TJ, UK
Phone: 44 1603 592186
Fax: 44 1603 592250
r.bowater@uea.ac.uk

References and Footnotes
  1. Wilkinson, Day & Bowater, Molec Microbiol. 40, 1241-1248 (2001).
  2. Lee, et al., EMBO J. 19, 1119-1129 (2000).
  3. Singleton, et al., Structure 7, 35-42 (1999).