Sse8387I, a useful eight base cutter for mammalian genome analysis (influence of methylation on the activity of Sse8387I).
To develop restriction enzymes that are useful for genome analysis, we previously performed screening and isolated Sse8387I from Streptomyces sp. strain 8387. Sse8387I is a restriction enzyme that recognizes 5'-CCTGCA/GG-3' and cleaves DNA at the site shown by the diagonal (Nucleic Acid Res., 18, 5637-5640). The present study evaluated the effects of methylation that is important when Sse8387I is used for genome analysis. Sse8387I lost cleavage activity after methylation of adenine or methylation of cytosine at any site in the recognition sequence. However, the recognition sequence of Sse8387I contains no CG sequence, which is the mammalian methylation sequence. In addition, we evaluated the effects of methylation of CG at sites other than the recognition sequence. The cleavage activity of Sse8387I was maintained even when CG sequences were present immediately before or after, or near the recognition sequence, and cytosine was methylated. These results suggest that CG methylation does not affect the cleavage activity of Sse8387I. Therefore, Sse8387I seems to be very useful for mammalian genome analysis.
Sagawa
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H
Genetic Engineering Section II, Biotechnology Research Laboratories, Takara Shuzo Co. Ltd, Shiga, Japan.
Ohshima
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A
Kato
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I
D044503:5-Methylcytosine; D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism*; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D008745:Methylation; D008969:Molecular Sequence Data; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
D044503:5-Methylcytosine; D000225:Adenine; D003596:Cytosine; D004279:DNA, Viral; D015257:DNA-Cytosine Methylases; D015252:Deoxyribonucleases, Type II Site-Specific; C081573:DNA modification methylase SssI; C074158:endodeoxyribonuclease Sse8387I; C024388:CTGCAG-specific type II deoxyribonucleases; C000730567:6-methyladenine