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Publication Abstract from PubMed

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.

Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis.,Yang W, Chen WY, Wang H, Ho JW, Huang JD, Woo PC, Lau SK, Yuen KY, Zhang Q, Zhou W, Bartlam M, Watt RM, Rao Z Nucleic Acids Res. 2011 Dec;39(22):9803-19. Epub 2011 Sep 5. PMID:21893587^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.