Structural and mutational analyses of the C-terminal domain of human APOBEC3G in complex with ssDNA

• Main Authors: I-Li Wang, 王顗棣
• Other Authors: Shwu-Huey Liaw
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/06353348164458865860

碩士 === 國立陽明大學 === 生命科學暨基因體科學研究所 === 98 === 　　　　To　date,　the　apolipoprotein　B　mRNA-editing　catalytic　proteins　(APOBEC)　including　APOBEC1-4　and　activation-induced　deaminase　(AID),　are　the　only　identified　C-to-U　editing　deaminases　involved　in　gene　diversity　and　antiviral　activity.　Only　the　primate　APOBEC3　genes　have　expanded　into　a　tandem　array　of　genes　termed　APOBEC3A-H.　Among　them,　APOBEC3G　is　able　to　restrict　the　infection　of　viral　infectivity-factor-defective　HIV-1　through　C-to-U　editing　of　the　viral　nascent　minus-strand　DNA.　This　protein　contains　two　putative　deaminase　domains,　in　which　the　C-terminal　domain　(APOBEC3GC)　is　responsible　for　the　deaminase　activity.　In　this　thesis,　the　wild-type　APOBEC3GC　recombinant　protein　was　first　expressed　and　isolated　successfully,　with　significant　deaminase　activity　on　a　variety　of　ssDNA.　After　extensive　screening　and　refinement　of　APOBEC3GC　with　a　14-mer　ssDNA,　some　crystals　were　grown　and　diffracted　x-ray　to　3　?痋@resolution.　Future　crystallization　refinement　and　structure　determination　are　under　investigation.　Recently,　several　structures　of　the　wild-type　and　mutant　APOBEC3GC　have　been　determined　by　NMR　and　X-ray　diffraction　methods.　These　structures　display　significant　differences　and　hence　distinct　ssDNA-binding　residues　are　predicted.　Particularly,　the　functional　roles　of　R215　and　R313　are　inconclusive.　These　two　arginine　residues　are　corresponding　to　R24　and　R112　in　AID,　and　the　R24W　and　R112C　mutants　are　the　hot　spots　in　the　hyper-IgM　patients.　The　R215W　and　R313C　mutants　were　then　generated　and　characterized.　Both　mutants　display　no　detectable　deaminase　activity.　R215W　exhibits　similar　circular　dichroism　spectra　compared　with　wild　type　APOBEC3GC.　The　ssDNA　binding　abilities　are　monitored　by　electrophoretic　mobility　shift　assay,　filter　binding　assay　and　isothermal　titration　calorimetry.　The　preliminary　data　suggest　that　the　replacement　of　R215　with　tryptophan　decreases　the　ssDNA　binding,　but　increase　the　Tm　value　by　~5　oC.