Zhu Zhenyu, Guan Shengxi, Robinson Derek, El Fezzazi Hanna, Quimby Aine, Xu Shuang-yong
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Sci Rep. 2014 Jan 23;4:3838. doi: 10.1038/srep03838.
Tth111II is a thermostable Type IIGS restriction enzyme that recognizes DNA sites CAARCA (R = A or G) and cleaves downstream at N11/N9. Here, the tth111IIRM gene was cloned and expressed in E. coli, and Tth111II was purified. The purified enzyme contains internally-bound S-adenosylmethionine (SAM). When the internal SAM was removed, the endonuclease activity was stimulated by adding SAM or its analog sinefungin. The cleavage intermediate is mostly top-strand nicked DNA on a single-site plasmid. Addition of duplex oligos with a cognate site stimulates cleavage activity of the one-site substrate. Tth111II cleaves a two-site plasmid DNA with equal efficiency regardless of site orientation. We propose the top-strand nicking is carried out by a Tth111II monomer and bottom-strand cleavage is carried out by a transient dimer. Tth111II methylates cleavage product-like duplex oligos CAAACAN9, but the modification rate is estimated to be much slower than the top-strand nicking rate. We cloned and sequenced a number of Tth111II star sites which are 1-bp different from the cognate sites. A biochemical pathway is proposed for the restriction and methylation activities of Tth111II.
Tth111II是一种热稳定的IIGS型限制性内切酶，它识别DNA位点CAARCA（R = A或G）并在下游N11/N9处切割。在此，tth111IIRM基因被克隆并在大肠杆菌中表达，然后对Tth111II进行了纯化。纯化后的酶含有内部结合的S-腺苷甲硫氨酸（SAM）。当去除内部SAM后，通过添加SAM或其类似物杀稻瘟菌素可刺激核酸内切酶活性。切割中间体主要是单一位点质粒上的上链切口DNA。添加具有同源位点的双链寡核苷酸可刺激单一位点底物的切割活性。Tth111II以相同效率切割双位点质粒DNA，而与位点方向无关。我们提出上链切口由Tth111II单体进行，而下链切割由瞬时二聚体进行。Tth111II使切割产物样双链寡核苷酸CAAACAN9甲基化，但估计修饰速率比上链切口速率慢得多。我们克隆并测序了一些与同源位点相差1个碱基对的Tth111II星号位点。针对Tth111II的限制和甲基化活性提出了一种生化途径。
