title: Organization of the BcgI restriction-modification protein for the cleavage of eight phosphodiester bonds in DNA.
abstract: Type IIB restriction-modification systems, such as BcgI, feature a single protein with both endonuclease and methyltransferase activities. Type IIB nucleases require two recognition sites and cut both strands on both sides of their unmodified sites. BcgI cuts all eight target phosphodiester bonds before dissociation. The BcgI protein contains A and B polypeptides in a 2:1 ratio: A has one catalytic centre for each activity; B recognizes the DNA. We show here that BcgI is organized as A(2)B protomers, with B at its centre, but that these protomers self-associate to assemblies containing several A(2)B units. Moreover, like the well known FokI nuclease, BcgI bound to its site has to recruit additional protomers before it can cut DNA. DNA-bound BcgI can alternatively be activated by excess A subunits, much like the activation of FokI by its catalytic domain. Eight A subunits, each with one centre for nuclease activity, are presumably needed to cut the eight bonds cleaved by BcgI. Its nuclease reaction may thus involve two A(2)B units, each bound to a recognition site, with two more A(2)B units bridging the complexes by protein-protein interactions between the nuclease domains.
authors.affiliation: The DNA-proteins Interaction Unit, School of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, UK.
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Smith
authors.forename: Jacqueline J T
authors.initials: JJ
authors.lastname: Marshall
authors.forename: Alistair J
authors.initials: AJ
authors.lastname: Jacklin
authors.forename: Susan E
authors.initials: SE
authors.lastname: Retter
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Halford
authors.forename: Frank
authors.initials: F
authors.lastname: Sobott
mesh_terms: D004247:DNA / Q000378:metabolism; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry; D021122:Protein Subunits / Q000737:chemistry; D014461:Ultracentrifugation
chemical_list: D021122:Protein Subunits; D004247:DNA; C073736:endodeoxyribonuclease BcgI; D015252:Deoxyribonucleases, Type II Site-Specific