Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination 英文参考文献.docVIP

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Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination 英文参考文献.doc

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Mutational Analysis of Highly Conserved Residues in the Phage PhiC31 Integrase Reveals Key Amino Acids Necessary for the DNA Recombination 英文参考文献

MutationalAnalysisofHighlyConservedResiduesinthe PhagePhiC31IntegraseRevealsKeyAminoAcids NecessaryfortheDNARecombination ShaohuiLiu1,JinfangMa1,WeiWang1,MaoxiangZhang1,QingtingXin1,SimanPeng1,RongxiuLi2, HuanzhangZhu1* 1State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, China, 2MOE Key Laboratory of Microbial Metabolism,andSchoolofLifeScienceandBiotechnology,ShanghaiJiaoTongUniversity,Shanghai,China Abstract Background: Amino acid sequence alignment of phage phiC31 integrase with the serine recombinases family revealed highlyconservedregionsoutsidethecatalyticdomain.Untilnow,nosystemmutationalorbiochemicalstudieshavebeen carriedouttoassesstherolesoftheseconservedresiduesintherecombinatonofphiC31integrase. Methodology/Principal Findings: To determine the functional roles of these conserved residues, a series of conserved residues were targeted by site-directed mutagenesis. Out of the 17 mutants, 11 mutants showed impaired or no recombinationability,asanalyzedbyrecombinationassaybothinvivoandinvitro.ResultsofDNAbindingactivityassays showedthatmutants(R18A,I141A,L143A,E153A,I432AandV571A)exhibitedagreatdecreaseinDNAbindingaffinity,and mutants(G182A/F183A,C374A,C376A/G377A,Y393AandV566A)hadcompletelylosttheirabilitytobindtothespecific targetDNAattBascomparedwithwild-typeprotein.Furtheranalysisofmutants(R18A,I141A,L143AandE153A)synapse andcleavageshowedthatthesemutantswereblockedinrecombinationatthestageofstrandcleavage. Conclusions/Significance: This data reveals that some of the highly conserved residues both in the N-terminus and C- terminusregionofphiC31integrase,playvitalrolesinthesubstratebindingandcleavage.Thecysteine-richmotifandthe C-tailval-richregionofphiC31integrasemayrepresentthemajorDNAbindingdomainsofphiC31integrase. Citation:LiuS,MaJ,WangW,ZhangM,XinQ,etal.(2010)MutationalAnalysisofHighlyConservedResiduesinthePhagePhiC31IntegraseRevealsKeyAmino AcidsNecessaryfortheDNARecombination.PL