Characterization of cleavage intermediate and star sites of RM.Tth111II.
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.
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Zhenyu
Z
Zhu
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Shengxi
S
Guan
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Derek
D
Robinson
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Hanna
H
El Fezzazi
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Aine
A
Quimby
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
Shuang-yong
SY
Xu
D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D004247:DNA; C027716:endodeoxyribonuclease Tth111II; D015252:Deoxyribonucleases, Type II Site-Specific