Characterization of cleavage intermediate and star sites of RM.Tth111II
Zhu, Z; Guan, S; Robinson, D; El Fezzazi, H; Quimby, A; Xu, SY
| HERO ID | 3539684 |
|---|---|
| In Press | No |
| Year | 2014 |
| Title | Characterization of cleavage intermediate and star sites of RM.Tth111II |
| Authors | Zhu, Z; Guan, S; Robinson, D; El Fezzazi, H; Quimby, A; Xu, SY |
| Journal | Scientific Reports |
| Volume | 4 |
| Page Numbers | 3838 |
| Abstract | 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. |
| Doi | 10.1038/srep03838 |
| Pmid | 24452415 |
| Wosid | WOS:000330045200005 |
| Is Certified Translation | No |
| Dupe Override | No |
| Is Public | Yes |
| Language Text | English |