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Article: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis
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TitleStructural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis
 
AuthorsYang, W2
Chen, WY3
Wang, H2 4
Ho, JWS5
Huang, JD1
Woo, PCY1
Lau, SKP1
Yuen, KY1
Zhang, Q2
Zhou, W2
Bartlam, M2
Watt, RM3
Rao, Z2 4
 
Issue Date2011
 
PublisherOxford University Press. The Journal's web site is located at http://nar.oxfordjournals.org/
 
CitationNucleic Acids Research, 2011, v. 39 n. 22, p. 9803-9819 [How to Cite?]
DOI: http://dx.doi.org/10.1093/nar/gkr660
 
AbstractAlkaline 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 β-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, revealing a 'doughnut-shaped' toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. 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. © The Author(s) 2011. Published by Oxford University Press.
 
DescriptionEpub ahead of print; Open Access Journal
 
ISSN0305-1048
2012 Impact Factor: 8.278
2012 SCImago Journal Rankings: 5.125
 
DOIhttp://dx.doi.org/10.1093/nar/gkr660
 
PubMed Central IDPMC3239189
 
ISI Accession Number IDWOS:000298186000035
Funding AgencyGrant Number
Ministry of Science and Technology of China2007CB914301
Research Grants Council of Hong Kong779109
University of Hong Kong
Funding Information:

Ministry of Science and Technology of China Project 973 (grant number 2007CB914301 to M.B.); General Research Fund (GRF) award from the Research Grants Council of Hong Kong (grant number 779109 to R.M.W.); Infection and Immunology Strategic Research Theme of the University of Hong Kong (to R.M.W.). Funding for open access charge: Ministry of Science and Technology of China Project 973 (grant number 2007CB914301 to M.B.).

 
ReferencesReferences in Scopus
 
DC FieldValue
dc.contributor.authorYang, W
 
dc.contributor.authorChen, WY
 
dc.contributor.authorWang, H
 
dc.contributor.authorHo, JWS
 
dc.contributor.authorHuang, JD
 
dc.contributor.authorWoo, PCY
 
dc.contributor.authorLau, SKP
 
dc.contributor.authorYuen, KY
 
dc.contributor.authorZhang, Q
 
dc.contributor.authorZhou, W
 
dc.contributor.authorBartlam, M
 
dc.contributor.authorWatt, RM
 
dc.contributor.authorRao, Z
 
dc.date.accessioned2011-09-23T05:41:43Z
 
dc.date.available2011-09-23T05:41:43Z
 
dc.date.issued2011
 
dc.description.abstractAlkaline 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 β-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, revealing a 'doughnut-shaped' toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. 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. © The Author(s) 2011. Published by Oxford University Press.
 
dc.description.naturepublished_or_final_version
 
dc.descriptionEpub ahead of print; Open Access Journal
 
dc.identifier.citationNucleic Acids Research, 2011, v. 39 n. 22, p. 9803-9819 [How to Cite?]
DOI: http://dx.doi.org/10.1093/nar/gkr660
 
dc.identifier.doihttp://dx.doi.org/10.1093/nar/gkr660
 
dc.identifier.eissn1362-4962
 
dc.identifier.epage9819
 
dc.identifier.hkuros195552
 
dc.identifier.isiWOS:000298186000035
Funding AgencyGrant Number
Ministry of Science and Technology of China2007CB914301
Research Grants Council of Hong Kong779109
University of Hong Kong
Funding Information:

Ministry of Science and Technology of China Project 973 (grant number 2007CB914301 to M.B.); General Research Fund (GRF) award from the Research Grants Council of Hong Kong (grant number 779109 to R.M.W.); Infection and Immunology Strategic Research Theme of the University of Hong Kong (to R.M.W.). Funding for open access charge: Ministry of Science and Technology of China Project 973 (grant number 2007CB914301 to M.B.).

 
dc.identifier.issn0305-1048
2012 Impact Factor: 8.278
2012 SCImago Journal Rankings: 5.125
 
dc.identifier.issue22
 
dc.identifier.openurl
 
dc.identifier.pmcidPMC3239189
 
dc.identifier.pmid21893587
 
dc.identifier.scopuseid_2-s2.0-83755224379
 
dc.identifier.spage9803
 
dc.identifier.urihttp://hdl.handle.net/10722/138889
 
dc.identifier.volume39
 
dc.languageeng
 
dc.publisherOxford University Press. The Journal's web site is located at http://nar.oxfordjournals.org/
 
dc.publisher.placeUnited Kingdom
 
dc.relation.ispartofNucleic Acids Research
 
dc.relation.referencesReferences in Scopus
 
dc.titleStructural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis
 
dc.typeArticle
 
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<description.abstract>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 &#946;-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&#8242;-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg 2+ or Mn 2+ ions. 5&#8242;-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9, revealing a &apos;doughnut-shaped&apos; toroidal trimeric arrangement with a central tapered channel, analogous to that of &#955;-exonuclease (Exo) from bacteriophage-&#955;. 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. &#169; The Author(s) 2011. Published by Oxford University Press.</description.abstract>
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Author Affiliations
  1. The University of Hong Kong Li Ka Shing Faculty of Medicine
  2. Nankai University
  3. Prince Philip Dental Hospital
  4. Tsinghua University
  5. Chinese University of Hong Kong