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Article: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

TitleFunctional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae
Authors
Issue Date2011
PublisherBioMed Central Ltd. The Journal's web site is located at http://www.biomedcentral.com/bmcmolbiol/
Citation
Bmc Molecular Biology, 2011, p. 16 How to Cite?
AbstractBackground: SXT is an integrating conjugative element (ICE) originally isolated from Vibrio cholerae, the bacterial pathogen that causes cholera. It houses multiple antibiotic and heavy metal resistance genes on its ca. 100kb circular double stranded DNA (dsDNA) genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. Here, we characterize the activities of an alkaline exonuclease (S066, SXT-Exo) and single strand annealing protein (S065, SXT-Bet) encoded on the SXT genetic element, which share significant sequence homology with Exo and Bet from bacteriophage lambda, respectively. Results: SXT-Exo has the ability to degrade both linear dsDNA and single stranded DNA (ssDNA) molecules, but has no detectable endonuclease or nicking activities. Adopting a stable trimeric arrangement in solution, the exonuclease activities of SXT-Exo are optimal at pH 8.2 and essentially require Mn2+ or Mg2+ ions. Similar to lambda-Exo, SXT-Exo hydrolyzes dsDNA with 5'- to 3'-polarity in a highly processive manner, and digests DNA substrates with 5'-phosphorylated termini significantly more effectively than those lacking 5'-phosphate groups. Notably, the dsDNA exonuclease activities of both SXT-Exo and lambda-Exo are stimulated by the addition of lambda-Bet, SXT-Bet or a single strand DNA binding protein encoded on the SXT genetic element (S064, SXT-Ssb). When co-expressed in E. coli cells, SXT-Bet and SXT-Exo mediate homologous recombination between a PCR-generated dsDNA fragment and the chromosome, analogous to RecET and lambda-Bet/Exo. Conclusions: The activities of the SXT-Exo protein are consistent with it having the ability to resect the ends of linearized dsDNA molecules, forming partially ssDNA substrates for the partnering SXT-Bet single strand annealing protein. As such, SXT-Exo and SXT-Bet may function together to repair or process SXT genetic elements within infected V. cholerae cells, through facilitating homologous DNA recombination events. The results presented here significantly extend our general understanding of the properties and activities of alkaline exonuclease and single strand annealing proteins of viral/bacteriophage origin, and will assist the rational development of bacterial recombineering systems.
Persistent Identifierhttp://hdl.handle.net/10722/134343
ISSN
2015 Impact Factor: 2.5
2015 SCImago Journal Rankings: 1.224
PubMed Central ID
ISI Accession Number ID
Funding AgencyGrant Number
Research Grants Council of Hong Kong779109
Chinese University of Hong Kong
Funding Information:

This work was supported by the Research Grants Council of Hong Kong through a GRF award (#779109) to RMW. WYC was supported by a Graduate Assistantship from The Chinese University of Hong Kong. We thank Prof. A. Francis Stewart for generously providing the pBADET gamma plasmid, and thank Prof. Matthew Waldor and Dr. John Beaber for providing the pJB1 plasmid, and for their insightful comments.

 

DC FieldValueLanguage
dc.contributor.authorChen, WYen_HK
dc.contributor.authorHo, JWSen_HK
dc.contributor.authorHuang, JDen_HK
dc.contributor.authorWatt, RMen_HK
dc.date.accessioned2011-06-17T09:18:05Z-
dc.date.available2011-06-17T09:18:05Z-
dc.date.issued2011en_HK
dc.identifier.citationBmc Molecular Biology, 2011, p. 16en_HK
dc.identifier.issn1471-2199en_HK
dc.identifier.urihttp://hdl.handle.net/10722/134343-
dc.description.abstractBackground: SXT is an integrating conjugative element (ICE) originally isolated from Vibrio cholerae, the bacterial pathogen that causes cholera. It houses multiple antibiotic and heavy metal resistance genes on its ca. 100kb circular double stranded DNA (dsDNA) genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. Here, we characterize the activities of an alkaline exonuclease (S066, SXT-Exo) and single strand annealing protein (S065, SXT-Bet) encoded on the SXT genetic element, which share significant sequence homology with Exo and Bet from bacteriophage lambda, respectively. Results: SXT-Exo has the ability to degrade both linear dsDNA and single stranded DNA (ssDNA) molecules, but has no detectable endonuclease or nicking activities. Adopting a stable trimeric arrangement in solution, the exonuclease activities of SXT-Exo are optimal at pH 8.2 and essentially require Mn2+ or Mg2+ ions. Similar to lambda-Exo, SXT-Exo hydrolyzes dsDNA with 5'- to 3'-polarity in a highly processive manner, and digests DNA substrates with 5'-phosphorylated termini significantly more effectively than those lacking 5'-phosphate groups. Notably, the dsDNA exonuclease activities of both SXT-Exo and lambda-Exo are stimulated by the addition of lambda-Bet, SXT-Bet or a single strand DNA binding protein encoded on the SXT genetic element (S064, SXT-Ssb). When co-expressed in E. coli cells, SXT-Bet and SXT-Exo mediate homologous recombination between a PCR-generated dsDNA fragment and the chromosome, analogous to RecET and lambda-Bet/Exo. Conclusions: The activities of the SXT-Exo protein are consistent with it having the ability to resect the ends of linearized dsDNA molecules, forming partially ssDNA substrates for the partnering SXT-Bet single strand annealing protein. As such, SXT-Exo and SXT-Bet may function together to repair or process SXT genetic elements within infected V. cholerae cells, through facilitating homologous DNA recombination events. The results presented here significantly extend our general understanding of the properties and activities of alkaline exonuclease and single strand annealing proteins of viral/bacteriophage origin, and will assist the rational development of bacterial recombineering systems.en_HK
dc.languageengen_US
dc.publisherBioMed Central Ltd. The Journal's web site is located at http://www.biomedcentral.com/bmcmolbiol/en_HK
dc.relation.ispartofBMC Molecular Biologyen_HK
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.rightsB M C Molecular Biology. Copyright © BioMed Central Ltd.-
dc.subject.meshBacterial Proteins - genetics - metabolism-
dc.subject.meshDNA, Bacterial - genetics - metabolism-
dc.subject.meshEnzyme Activation - drug effects-
dc.subject.meshExodeoxyribonucleases - antagonists and inhibitors - metabolism-
dc.subject.meshVibrio cholerae - enzymology - genetics - metabolism-
dc.titleFunctional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio choleraeen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=1471-2199&volume=12, article no. 16&spage=&epage=&date=2011&atitle=Functional+characterization+of+an+alkaline+exonuclease+and+single+strand+annealing+protein+from+the+SXT+genetic+element+of+Vibrio+choleraeen_US
dc.identifier.emailChen, WY: chenwy@hku.hken_HK
dc.identifier.emailWatt, RM: rmwatt@hku.hken_HK
dc.identifier.authorityChen, WY=rp01487en_HK
dc.identifier.authorityWatt, RM=rp00043en_HK
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.1186/1471-2199-12-16en_HK
dc.identifier.pmid21501469-
dc.identifier.pmcidPMC3118119-
dc.identifier.scopuseid_2-s2.0-79955100014en_HK
dc.identifier.hkuros185797en_US
dc.identifier.volume12, article no. 16en_US
dc.identifier.spage16en_HK
dc.identifier.epage16en_HK
dc.identifier.eissn1471-2199-
dc.identifier.isiWOS:000291746300001-
dc.publisher.placeUnited Kingdomen_HK
dc.identifier.scopusauthoridChen, WY=37100973400en_HK
dc.identifier.scopusauthoridHo, JWS=7402650205en_HK
dc.identifier.scopusauthoridHuang, JD=37101681500en_HK
dc.identifier.scopusauthoridWatt, RM=7102907536en_HK
dc.identifier.citeulike9176662-

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