title: C-terminal deletion mutants of the FokI restriction endonuclease.
abstract: We have constructed two C-terminal deletion mutants of the FokI restriction endonuclease by using the polymerase-chain-reaction technique and expressed them in Escherichia coli. The two mutant proteins (MP) of 41 and 30 kDa, were purified to homogeneity and their DNA-binding properties were characterized. The 41-kDa MP specifically binds the DNA sequence, 5'-GGATG/3'-CCTAC, like the wild-type (wt) FokI, but does not cleave DNA. The 30-kDa MP does not bind DNA. The affinity of the 41-kDa MP for the DNA substrate is comparable to that of wt FokI. The 41-kDa MP interacts with its substrate like the wt FokI, as revealed by hydroxyl radical footprinting experiments. In the presence of a DNA substrate, the 41-kDa MP is cleaved by trypsin into a 30-kDa N-terminal fragment and an 11-kDa C-terminal fragment. Addition of the HPLC-purified 11-kDa C-terminal fragment to the 30-kDa MP restores its sequence-specific DNA-binding property. These results confirm that the N-terminal 41-kDa fragment of the FokI ENase constitutes the DNA recognition domain of the ENase.
authors.affiliation: Department of Environmental Health Sciences, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205.
authors.forename: L
authors.initials: L
authors.lastname: Li
authors.forename: L P
authors.initials: LP
authors.lastname: Wu
authors.forename: R
authors.initials: R
authors.lastname: Clarke
authors.forename: S
authors.initials: S
authors.lastname: Chandrasegaran
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D017353:Gene Deletion; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D013379:Substrate Specificity; D014357:Trypsin
chemical_list: D004269:DNA, Bacterial; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific; D014357:Trypsin