Intermolecular V(D)J recombination.

V(D)J recombination plays a prominent role in the generation of the antigen receptor repertoires of B and T lymphocytes. It is also likely to be involved in the formation of chromosomal translocations, some of which may result from interchromosomal recombination. We have investigated the potential of the V(D)J recombination machinery to perform intermolecular recombination between two plasmids, either unlinked or linked by catenation. In either case, recombination occurs in trans to yield signal and coding joints, and the results do not support the existence of a mechanistic block to the formation of coding joints in trans. Instead, we observe that linearization of the substrate, which does not alter the cis or trans status of the recombination signals, causes a specific and dramatic reduction in coding joint formation. This unexpected result leads us to propose a "release and recapture" model for V(D)J recombination in which coding ends are frequently released from the postcleavage complex and the efficiency of coding joint formation is influenced by the efficiency with which such ends are recaptured by the complex. This implies the existence of mechanisms, operative during recombination of chromosomal substrates, that act to prevent coding end release or to facilitate coding end recapture.

Tumor suppressor p16INK4A: determination of solution structure and analyses of its interaction with cyclin-dependent kinase 4.

The solution structure of the tumor suppressor p16INK4A has been determined by NMR, and important recognition regions of both cdk4 and p16INK4A have been identified. The tertiary structure of p16INK4A contains four helix-turn-helix motifs linked by three loops. Twelve tumorigenic mutants of p16INK4A have been constructed and analyzed for their structure and activity, and new mutants have been designed rationally. A fragment of 58 residues at the N terminus of cdk4 important for p16INK4A binding has been identified. The importance of this region was further verified by mutational analysis of cdk4. These results and docking experiments have been used to assess possible modes of binding between p16INK4A and cdk4.

Tumor suppressor p16INK4A: structural characterization of wild-type and mutant proteins by NMR and circular dichroism.

The tumor suppressor p16INK4A with eight N-terminal amino acids deleted (p16/delta 1-8) was expressed in Escherichia coli without any fusion artifacts and purified. The integrity of p16/delta 1-8 was confirmed by mass spectrometry, and its activity was demonstrated by in vitro cdk4 inhibition assay. Various physical methods were used to characterize the molecular and structural properties of p16/delta 1-8. The protein was found to oligomerize in vitro, as demonstrated by gel electrophoresis, mass spectrometry, and NMR. Various approaches, including changes of concentration and pH, additions of salts, detergents, and various organic solvents, and construction of a C-terminal deletion mutant and a cysteine mutant were used to try to reduce the extent of oligomerization. Only decreasing the protein concentration was found to reduce oligomerization. The affinity between p16 molecules in vivo was demonstrated by the yeast two-hybrid system. The protein was found to be very unstable on the basis of urea- and guanidinium chloride-induced denaturation studies monitored by NMR and CD, respectively. Despite these unfavorable properties, total NMR assignments were accomplished with uniform 13C and 15N isotope labeling. All multidimensional NMR experiments were performed at a very low concentration of 0.2 mM. The secondary structure was then determined from the NMR data. The results of NMR and CD studies indicate that the protein is highly alpha-helical, and the ankyrin repeat sequences show helix-turn-helix structures. This is the first structural information obtained for the important motif of ankyrin repeats. Overall, p16/delta 1-8 appears to be conformationally flexible. In order to understand the structural basis of the functional changes for some mutants existing in tumor cells, several missense mutants of p16/delta 1-8 were constructed. Four of them were expressed at high levels and purified. The molecular and structural properties of these mutants were analyzed by CD and NMR and compared with the corresponding properties of wild-type p16/delta 1-8. The results suggest that the functional changes in P114L and G101W are likely to be related to global conformational changes. In addition, we have demonstrated that the tendency of aggregation increases significantly by a single D84H mutation.

Synthesis, cloning and expression of a synthetic gene for high potential iron protein from Chromatium vinosum.

A synthetic gene encoding the peptide sequence for the low molecular weight (M(r) approximately 9600 Da) high-potential iron protein (HiPIP) from the photosynthetic bacterium Chromatium vinosum has been constructed by shotgun ligation of twelve complimentary oligonucleotides varying in size from 42-mers to 48-mers. After cloning the gene into a pET-21d(+) vector, expression of holoprotein in yields of 35 mg/liter of culture was obtained following induction with isopropyl-beta-D-thiogalactoside (IPTG). The recombinant protein was characterized by electronic absorption, 1H NMR, electrochemistry, N-terminal sequencing and amino acid analysis. This is the first example of the expression of a high potential ferredoxin containing a fully constituted [Fe4S4] cluster.