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Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

Author:
Yang, Wen  Chen, Wen-yang  Wang, Hui  Ho, John W. S.  Huang, Jian-Dong  Woo, Patrick C. Y.  Lau, Susanna K. P.  Yuen, Kwok-Yung  Zhang, Qionglin  Zhou, Weihong  Bartlam, Mark  Watt, Rory M.  Rao, Zihe  


Journal:
NUCLEIC ACIDS RESEARCH


Issue Date:
2011


Abstract(summary):

Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced beta-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5'-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg(2+) or Mn(2+) ions. 5'-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 A, revealing a 'doughnut-shaped' toroidal trimeric arrangement with a central tapered channel, analogous to that of lambda-exonuclease (Exo) from bacteriophage-lambda. Active sites containing two bound Mg(2+) ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases.


Page:
9803---9819


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