Restriction/modification polypeptides, polynucleotides, and methods

摘要

The present invention relates to the discovery of a novel restriction/modification system in Caldicellulosiruptor bescii. The discovered restriction enzyme is a HaeIII-like restriction enzyme that possesses a thermophilic activity profile. The restriction/modification system also includes a methyltransferase, M.CbeI, that methylates at least one cytosine residue in the CbeI recognition sequence to m4C. Thus, the invention provides, in various aspects, isolated CbeI or M.CbeI polypeptides, or biologically active fragments thereof; isolated polynucleotides that encode the CbeI or M.CbeI polypeptides or biologically active fragments thereof, including expression vectors that include such polynucleotide sequences; methods of digesting DNA using a CbeI polypeptide; methods of treating a DNA molecule using a M.CbeI polypeptide; and methods of transforming a Caldicellulosiruptor cell.

主权项

1. A method of transforming a microbial cell, the method comprising:
treating a DNA molecule comprising at least one 5′-GGCC-3′ sequence with a M.CbeI polypeptide under condition effective for the M.CbeI polypeptide to methylate at least one C residue of the 5′-GGCC-3′ sequence; introducing the methylated polynucleotide into the microbial cell that comprises a thermophile or a hyperthermophile.