The　bacterial　restriction-modification　system　(RMS)　plays　a　pivotal　role　in　cellular　defense　against　foreign　DNA.　It　can　be　classified　into　four　major　groups:　types　I,　II,　III　and　IV.　Asc　I　belongs　to　the　type　II　restriction　endonucleases　and　can　specifically　recognize　an　8-bp　DNA　element.　Despite　being　used　extensively　in　molecular　cloning,　the　information　for　protein　expression,　purification　and　three　dimensional　structure　of　Asc　I　has　been　lacking.　Here,　we　developed　a　bacterial　expression　system　and　an　efficient　purification　procedure,　and　obtained　recombinant　Asc　I　protein　with　a　yield　of　~　2.　5　mg　per　liter　culture　for　over　95%　purity.　Further　enzymatic　characterization　showed　that　the　optimum　temperature　and　for　enzymatic　reaction　were　37oC　and　pH　7.　5~8.　5,　respectively.　The　enzyme　required　divalent　metal　ions　for　activity,　with　a　strong　preference　for　Mg2+　and　Mn2+.　Furthermore,　with　the　Small　Angle　X-ray　Scattering　(SAXS)　and　extensive　mutagenesis　analyses,　we　constructed　structural　models　showing　how　Asc　I　might　interact　with　its　DNA　substrate　in　solution.　Together,　our　work　provides　biochemical　and　structural　characterizations　of　Asc　I　endonuclease,　and　paves　the　way　for　further　applications　of　the　enzyme　in　molecular　cloning.　©　Editorial　Office　of　Acta　Horticulturae　Sinica.　All　rights　reserved.