Sequence Specificity of BAL 31 Nuclease for ssDNA Revealed by Synthetic Oligomer Substrates Containing Homopolymeric Guanine Tracts 英文参考文献.docVIP

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Sequence Specificity of BAL 31 Nuclease for ssDNA Revealed by Synthetic Oligomer Substrates Containing Homopolymeric Guanine Tracts 英文参考文献.doc

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Sequence Specificity of BAL 31 Nuclease for ssDNA Revealed by Synthetic Oligomer Substrates Containing Homopolymeric Guanine Tracts 英文参考文献

SequenceSpecificityofBAL31NucleaseforssDNA RevealedbySyntheticOligomerSubstratesContaining HomopolymericGuanineTracts AprilMarrone1,2,JackBallantyne1,2 * 1Graduate Program in Chemistry, Department of Chemistry, University of Central Florida, Orlando, Florida, United States of America, 2National Center for Forensic Science,Orlando,Florida,UnitedStatesofAmerica Abstract Background:TheextracellularnucleasefromAlteromonasespejiana,BAL31catalyzesthedegradationofsingle-stranded andlinearduplexDNAto59-mononucleotides,cleavesnegativelysupercoiledDNAtothelinearduplexform,andcleaves duplexDNAinresponsetothepresenceofapurinicsites. Principal Findings: In this work we demonstrate that BAL 31 activity is affected by the presence of guanine in single- stranded DNA oligomers. Specifically, nuclease activity is shown to be affected by guanine’s presence in minimal homopolymerictractsinthemiddleofshortoligomersubstratesandalsobyitspresenceatthe39endoftenandtwenty baseoligomers.GNCrichregionsindsDNAareknowntocauseadecreaseintheenzyme’snucleaseactivitywhichhasbeen attributedtotheincreasedthermalstabilityoftheseregions,thusmakingitmoredifficulttounwindthestrandsrequired forenzymeaccess.Ourresultsindicatethatanadditionalphenomenoncouldbewhollyorpartlyresponsibleforthelossof activityintheseGNCrichregions.Thusthepresenceofaguaninetractperseimpairstheenzyme’sfunctionality,possibly duetothetract’sbulkynatureandpreventingefficientprogressionthroughtheactivesite. Conclusions: This study has revealed that the general purpose BAL 31 nuclease commonly used in molecular genetics exhibits a hithertofore non-characterized degree of substrate specificity with respect to single-stranded DNA (ssDNA) oligomers.Specifically,BAL31nucleaseactivitywasfoundtobeaffectedbythepresenceofguanineinssDNAoligomers. Citation: Marrone A, Ballantyne J (2008) Sequence Specificity of BAL 31 Nuclease for ssDNA Revealed by Synthetic Oligomer Substrates Containing HomopolymericGuanineTracts.PLoSONE3(10):e3595.doi:10.1371