We have found that mutations in the gene daf-2 can cause fertile, active, adult Caenorhabditis elegans hermaphrodites to live more than twice as long as wild type. This lifespan extension, the largest yet reported in any organism, requires the activity of a second gene, daf-16. Both genes also regulate formation of the dauer larva, a developmentally arrested larval form that is induced by crowding and starvation and is very long-lived. Our findings raise the possibility that the longevity of the dauer is not simply a consequence of its arrested growth, but instead results from a regulated lifespan extension mechanism that can be uncoupled from other aspects of dauer formation. daf-2 and daf-16 provide entry points into understanding how lifespan can be extended.

The URS2 region of the Saccharomyces cerevisiae HO upstream region contains 10 binding sites for the Swi4p/Swi6p transcription factor and confers Swi4p dependence for transcription. Using a hybrid promoter, UAS GAL (upstream activation sequence of GAL1)-URS2R, in which the GAL1-10 regulatory region is fused to the proximal 360 bp of URS2, we isolated mutants in which Swi4p is no longer required for transcription. Mutations of SIN4, ROX3, SRB8, SRB9, SRB10, SRB11, and two novel genes, NUT1 and NUT2, relieve the requirement of Swi4p for expression of this reporter. We found that NUT1 (open reading frame [ORF] YGL151w) is a nonessential gene, that NUT2 (ORF YPR168w) is essential, and that both Nut1p and Nut2p encode nuclear proteins. Deletion of NUT1 causes a constitutive, Swi4p-independent phenotype only in combination with the nut2-1 allele or an allele of CCR4. In contrast, inactivation of a temperature-sensitive allele of NUT2, nut2-ts70, alone causes constitutivity. nut1⌬ nut2-1 cells and sin4⌬ cells exhibit Swi4p-independent expression of an ho-lacZ reporter but not of an intact ho gene. Likewise, a pPHO5-lacZ construct is constitutively expressed in nut1 nut2 mutants relative to their wild-type counterparts. These results suggest that Nut1p, Nut2p, Sin4p, and Ccr4p define a group of proteins that negatively regulate transcription in a subtle manner which is revealed by artificial reporter genes.

We designed a single-chain variant of the Arc repressor homodimer in which the ␤ strands that contact operator DNA are connected by a hairpin turn and the ␣ helices that form the tetrahelical scaffold of the dimer are attached by a short linker. The designed protein represents a noncyclic permutation of secondary structural elements in another single-chain Arc molecule (Arc-L1-Arc), in which the two subunits are fused by a single linker. The permuted protein binds operator DNA with nanomolar affinity, refolds on the submillisecond time scale, and is as stable as Arc-L1-Arc. The crystal structure of the permuted protein reveals an essentially wild-type fold, demonstrating that crucial folding information is not encoded in the wild-type order of secondary structure. Noncyclic rearrangement of secondary structure may allow grouping of critical activesite residues in other proteins and could be a useful tool for protein design and minimization. circular permutation ͉ protein folding ͉ protein structure I magine a protein in which the ␣-helices and ␤-strands are disconnected but otherwise arranged properly in three dimensions, so that the hydrophobic core and most native interactions remain intact. There are probably many ways to connect the secondary-structure elements that are compatible with stable folding. For example, cyclic permutation of secondary structure (Fig. 1A) is tolerated in numerous proteins and occurs naturally in some protein families (1-10). After cyclic permutation, a wild-type block of N-terminal sequence is shifted to the C terminus of the permuted protein. Threedimensional domain swapping, in which a monomeric protein fold is reproduced with structural parts donated by different subunits in a multimer (Fig. 1B), provides another instance of rearranged structural elements (11,12). These examples suggest that the essential feature of a protein fold is the complementary packing of secondary structural elements and not the precise manner in which these elements are connected. If this model is correct, then many permutations of protein structural elements should be allowed. However, to our knowledge, no successful examples of noncyclic permutations of protein structural elements have been reported.Arc repressor from bacteriophage P22 is a small transcription factor that folds as a symmetric homodimer. Two Arc dimers bind cooperatively to adjacent subsites in arc operator DNA (13-16). Each Arc subunit consists of a ␤-strand and two ␣-helices (␣A and ␣B); the packing of these elements from both subunits of the dimer forms a single hydrophobic core. The ␤-strands pair to form an antiparallel ␤-sheet, which contacts the major groove of operator DNA. A f lexible N-terminal arm and residues near the beginning of helix B make other DNA contacts. Joining two Arc subunits with a 15-residue linker results in a single-chain molecule (Arc-L1-Arc) that is more stable than wild-type Arc and binds operator DNA at lower protein concentrations (17,18). The wild-type order of secondary structure is preserved in Arc-L...