ANALYSIS OF MOLECULAR STRUCTURE AND REACTION MECHANISM OF RESTRICTION ENDONUCLEASE FROM ACIDOPHILIC BACTERIA

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1) Cloning and sequencing of the retriction-modification system gene from acidophilic bacteria.The Afa22MI restriction-modification system from Acidocella facilis 22M recognizes the nucleotide sequence CGATCG.The M.Afa22MI gene was cloned into E.coli XL-1 Blue MRF^1 and the nucleotide sequence was determined. The homologous regions observed in 5-methyl cytosine methyltransferases (C5-MTase) were identified in the deduced amino acid sequence of M.Afa22MI.These conserved regions and the carboxyl terminal sequence of the M.Afa22MI showed high similarity with those of M.XorII which recognized same nucleotide sequence. The ORF located upstream of MTase gene showed high homology with the very short patch repair endonuclease of XorII and other vsr gene found in C5-MTase-endonuclease system.2) Purification and characterization of DNA methylases.Two DNA methyltransferases, M.Afa22MI and M.Afa16RI,were purified and characterized from same species Acidocella facilis 22M and 16R,respectively.3) Determination of methylation specificity of sequence-specific DNA methylase using MALDI-TOF MS.Time-dependent phosphodiesterase digestion of methylated oligonucleotide coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has enabled a sensitive and straightforward method for determing the methylation specificity of type II DNA methyltransferase. The mass deference between the peaks corresponded to the individual nucleotide released by the enzymatic cleavage, and the methylation site can be explicitly identified in reference to the know sequence of the substrate DNA.4) Crystallization of type II restriction endonuclease.We prepared large amounts of the homogeneous R.Aor 13HI,and crystallized it with an oligonucleotide containing it's recognition site (5'-TCCGGA-3') by hanging drop vapor diffusion method. A small crystal was obtained by precipitation with polyethylene glycol 4000 or 6000,100 mM HEPES (pH8.0), 100mM NaCl.