Lydia F. Dorner

Structure of the multimodular endonuclease FokI bound to DNA.

FokI is a member of an unusual class of bipartite restriction enzymes that recognize a specific DNA sequence and cleave DNA nonspecifically a short distance away from that sequence. Because of its unusual bipartite nature, FokI has been used to create artificial enzymes with new specificities. We have determined the crystal structure at 2.8 Å resolution of the complete FokI enzyme bound to DNA. As anticipated, the enzyme contains amino- and carboxy-terminal domains corresponding to the DNA-recognition and cleavage functions, respectively. The recognition domain is made of three smaller subdomains (D1, D2 and D3) which are evolutionarily related to the helix–turn–helix-containing DNA-binding domain of the catabolite gene activator protein CAP. The CAP core has been extensively embellished in the first two subdomains, whereas in the third subdomain it has been co-opted for protein–protein interactions. Surprisingly, the cleavage domain contains only a single catalytic centre, raising the question of how monomeric FokI manages to cleave both DNA strands. Unexpectedly, the cleavage domain is sequestered in a ‘piggyback’ fashion by the recognition domain. The structure suggests a new mechanism for nuclease activation and provides a framework for the design of chimaeric enzymes with altered specificities.

A MITOGEN-ACTIVATED PROTEIN KINASE PATHWAY IS REQUIRED FOR MU -OPIOID RECEPTOR DESENSITIZATION

The μ-opioid receptor mediates not only the beneficial painkilling effects of opiates like morphine but also the detrimental effects of chronic exposure such as tolerance and dependence. Different studies have linked tolerance to opioid receptor desensitization. Agonist activation of the μ-opioid receptor stimulates a mitogen-activated protein kinase (MAPK) activity, but the functional significance of this pathway remains unclear. We have focused on the MAPK signaling cascade to study μ-opioid receptor desensitization. We report that inhibition of the MAPK pathway blocks desensitization of μ-opioid receptor signaling as well as the loss of receptor density due to internalization. Our results suggest that a feedback signal emanating from the MAPK cascade is required for μ-opioid receptor desensitization.

Structure of restriction endonuclease BamHI phased at 1.95 å resolution by MAD analysis

BACKGROUND Type II restriction endonucleases recognize DNA sequences that vary between four to eight base pairs, and require only Mg2+ as a cofactor to catalyze the hydrolysis of DNA. Their protein sequences display a surprising lack of similarity, and no recurring structural motif analogous to the helix-turn-helix or the zinc finger of transcription factors, has yet been discovered. RESULTS We have determined the crystal structure of restriction endonuclease BamHI at 1.95 A resolution. The structure was solved by combining phase information derived from multi-wavelength X-ray data by algebraic and maximum likelihood methods. The BamHI subunit consists of a central beta-sheet with alpha-helices on both sides. The dimer configuration reveals a large cleft which could accommodate B-form DNA. Mutants of the enzyme that are deficient in cleavage are located at or near the putative DNA-binding cleft. BamHI and endonuclease EcoRI share a common core motif (CCM) consisting of five beta-strands and two helices. It remains to be determined if other restriction enzymes also contain the CCM. CONCLUSIONS The structure of BamHI provides the first clear evidence that there may be substantial structural homology amongst restriction enzymes, even though it is undetectable at the sequence level.

Sequence selectivity and degeneracy of a restriction endonuclease mediated by DNA intercalation.

The crystal structure of the HincII restriction endonuclease–DNA complex shows that degenerate specificity for blunt-ended cleavage at GTPyPuAC sequences arises from indirect readout of conformational preferences at the center pyrimidine-purine step. Protein-induced distortion of the DNA is accomplished by intercalation of glutamine side chains into the major groove on either side of the recognition site, generating bending by either tilt or roll at three distinct loci. The intercalated side chains propagate a concerted shift of all six target-site base pairs toward the minor groove, producing an unusual cross-strand purine stacking at the center pyrimidine–purine step. Comparison of the HincII and EcoRV cocrystal structures suggests that sequence-dependent differences in base–stacking free energies are a crucial underlying factor mediating protein recognition by indirect readout.

A view of consecutive binding events from structures of tetrameric endonuclease SfiI bound to DNA.

Many reactions in cells proceed via the sequestration of two DNA molecules in a synaptic complex. SfiI is a member of a growing family of restriction enzymes that can bind and cleave two DNA sites simultaneously. We present here the structures of tetrameric SfiI in complex with cognate DNA. The structures reveal two different binding states of SfiI: one with both DNA‐binding sites fully occupied and the other with fully and partially occupied sites. These two states provide details on how SfiI recognizes and cleaves its target DNA sites, and gives insight into sequential binding events. The SfiI recognition sequence (GGCCNNNN↓NGGCC) is a subset of the recognition sequence of BglI (GCCNNNN↓NGGC), and both enzymes cleave their target DNAs to leave 3‐base 3′ overhangs. We show that even though SfiI is a tetramer and BglI is a dimer, and there is little sequence similarity between the two enzymes, their modes of DNA recognition are unusually similar.

Cloning, analysis and expression of the HindIII R-M-encoding genes.

The genes encoding the HindIII restriction endonuclease (R.HindIII ENase) and methyltransferase (M.HindIII MTase) from Haemophilus influenzae Rd were cloned and expressed in Escherichia coli and their nucleotide (nt) sequences were determined. The genes are transcribed in the same orientation, with the ENase-encoding gene (hindIIIR) preceding the MTase-encoding gene (hindIIIM). The two genes overlap by several nt. The ENase is predicted to be 300 amino acids (aa) in length (34,950 Da); the MTase is predicted to be 309 aa (35,550 Da). The HindIII ENase and MTase activities increased approx. 20-fold when the genes were brought under the control of an inducible lambda pL promoter. Highly purified HindIII ENase and MTase proteins were prepared and their N-terminal aa sequences determined. In H. influenzae Rd, the HindIII R-M genes are located between the holC and valS genes; they are not closely linked to the HindII R-M genes.

Crystallization and preliminary X‐ray analysis of restriction endonuclease FokI bound to DNA

© 1997 Federation of European Biochemical Societies.