Resilin is a member of a family of elastic proteins that includes elastin, as well as gluten, gliadin, abductin and spider silks. Resilin is found in specialized regions of the cuticle of most insects, providing low stiffness, high strain and efficient energy storage; it is best known for its roles in insect flight and the remarkable jumping ability of fleas and spittle bugs. Previously, the Drosophila melanogaster CG15920 gene was tentatively identified as one encoding a resilin-like protein (pro-resilin). Here we report the cloning and expression of the first exon of the Drosophila CG15920 gene as a soluble protein in Escherichia coli. We show that this recombinant protein can be cast into a rubber-like biomaterial by rapid photochemical crosslinking. This observation validates the role of the putative elastic repeat motif in resilin function. The resilience (recovery after deformation) of crosslinked recombinant resilin was found to exceed that of unfilled synthetic polybutadiene, a high resilience rubber. We believe that our work will greatly facilitate structural investigations into the functional properties of resilin and shed light on more general aspects of the structure of elastomeric proteins. In addition, the ability to rapidly cast samples of this biomaterial may enable its use in situ for both industrial and biomedical applications.

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The interaction between DNA polymerases and sliding clamp proteins confers processivity in DNA synthesis. This interaction is critical for most DNA replication machines from viruses and prokaryotes to higher eukaryotes. T he replication of DNA in eubacteria involves many proteins organized into a complex multifunctional machine termed the replisome. A central enzyme is the multisubunit DNA polymerase III holoenzyme. In Escherichia coli, and probably in most other eubacteria, the DnaE ortholog (␣ subunit) is in the core of the replicative polymerase, whereas in many Grampositive organisms a related enzyme, PolC, is proposed to have this function (1). The processivity of the polymerase is conferred by the direct interaction of the ␤ subunit (clamp protein) of DNA polymerase III (2, 3), with the DnaE (and presumably PolC) subunits. ␤ is loaded onto DNA by a clamp loader comprised of single ␦ and ␦Ј subunits and four ͞␥ subunits (1). The ␤ dimer thence encircles the DNA without actually binding to it. In addition to DnaE, three other E. coli DNA polymerases appear to interact with ␤. PolB (DNA polymerase II) is involved in DNA repair (4) and the addition of ␤ and the clamp loader increases its processivity in vitro (5, 6). Similarly, ␤ and the clamp loader together increase both the processivity (7) and efficiency (8) of DNA synthesis by DNA polymerase IV (DinB). ␤ also appears to play a similar role in the activity of DNA polymerase V (8) (the UmuDЈ 2 UmuC complex) and the UmuD subunit has been shown to bind to ␤ (9).Experimental evidence shows that at least some ␤-binding proteins can interact productively with ␤ from heterologous species. For example, PolC subunits from Staphylococcus aureus, Streptococcus pyogenes, and Bacillus subtilis can use E. coli ␤ as their processivity subunit (1, 10, 11). In contrast, E. coli DnaE cannot use ␤ from the other species (11), the E. coli clamp loader complex cannot load S. aureus ␤ (11), and the S. pyogenes clamp loader complex cannot load E. coli ␤ (1).In the absence of any experimentally identified ␤-binding sites in proteins, a bioinformatics approach was undertaken to identify putative ␤-binding motifs. The role of the putative motif was then examined by yeast two-hybrid and peptide-binding experiments with native and modified sequences. Materials and MethodsSources of Amino Acid Sequences. Amino acid sequences and alignments were derived from: PSI-BLAST of the protein database at the National Center for Biotechnology Information (NCBI), and BLAST of preliminary sequence data from NCBI at http:͞͞ www.ncbi.nlm.nih.gov͞Microb_blast͞unfinishedgenome.html, Institute for Genomic Research at http:͞͞www.tigr.org, Department of Energy Joint Genome Institute at http:͞͞spider.jgipsf.org͞JGI_microbial͞html͞, Sanger Center at http:͞͞ www.sanger.ac.uk͞DataSearch͞omniblast.shtml, and ERGO at http:͞͞wit.integratedgenomics.com͞IGwit͞. Alignments of available sequences of all members of eubacterial protein families known to bind to ␤ were compiled with manual editing in regions of variab...

The epsilon subunit of the Escherichia coli replicative DNA polymerase III is the proofreading 3'-5' exonuclease. Structures of its catalytic N-terminal domain (epsilon186) were determined at two pH values (5.8 and 8.5) at resolutions of 1.7-1.8 A, in complex with two Mn(II) ions and a nucleotide product of its reaction, thymidine 5'-monophosphate. The protein structure is built around a core five-stranded beta sheet that is a common feature of members of the DnaQ superfamily. The structures were identical, except for differences in the way TMP and water molecules are coordinated to the binuclear metal center in the active site. These data are used to develop a mechanism for epsilon and to produce a plausible model of the complex of epsilon186 with DNA.