title: Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities.
abstract: The creation of restriction enzymes with programmable DNA-binding and -cleavage specificities has long been a goal of modern biology. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. However, a lack of structural information on Type IIL enzymes has limited the repertoire that can be rationally engineered. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation (5'-TCCRAC-3'; R = purine) and provides a molecular basis for changing specificity at four of the six base pairs of the recognition sequence (5'-TCCRAC-3'). Surprisingly, the enzyme is resilient to specificity changes at the first position of the recognition sequence (5'-TCCRAC-3'). Collectively, the structure provides a basis for engineering further derivatives of MmeI and delineates which base pairs of the recognition sequence are more amenable to alterations than others.
authors.affiliation: Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York, United States of America.
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Callahan
authors.affiliation: New England Biolabs Inc., Ipswich, Massachusetts, United States of America.
authors.forename: Yvette A
authors.initials: YA
authors.lastname: Luyten
authors.affiliation: Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York, United States of America.
authors.forename: Yogesh K
authors.initials: YK
authors.lastname: Gupta
authors.affiliation: New England Biolabs Inc., Ipswich, Massachusetts, United States of America.
authors.forename: Geoffrey G
authors.initials: GG
authors.lastname: Wilson
authors.affiliation: New England Biolabs Inc., Ipswich, Massachusetts, United States of America.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Roberts
authors.affiliation: New England Biolabs Inc., Ipswich, Massachusetts, United States of America.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Morgan
authors.affiliation: Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York, United States of America.
authors.forename: Aneel K
authors.initials: AK
authors.lastname: Aggarwal
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry*; D006868:Hydrolysis; D008969:Molecular Sequence Data
chemical_list: D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific