A large fragment of the dissimilatory sulfite reductase genes (dsrAB) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. In addition, the sequence of the dsrAB gene homologs of the sulfite reducer Desulfitobacterium dehalogenans was determined. In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny. For example, the DsrAB sequences of several Desulfotomaculum species (low G؉C gram-positive division) and two members of the genus Thermodesulfobacterium (a separate bacterial division) were monophyletic with ␦-proteobacterial DsrAB sequences. The most parsimonious interpretation of these data is that dsrAB genes from ancestors of as-yet-unrecognized sulfate reducers within the ␦-Proteobacteria were laterally transferred across divisions. A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of dsrAB genes. Evidence for a dsrAB lateral gene transfer event also was found within the ␦-Proteobacteria, affecting Desulfobacula toluolica. The root of the dsr tree was inferred to be within the Thermodesulfovibrio lineage by paralogous rooting of the alpha and beta subunits. This rooting suggests that the dsrAB genes in Archaeoglobus species also are the result of an ancient lateral transfer from a bacterial donor. Although these findings complicate the use of dsrAB genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.Siroheme dissimilatory sulfite reductases (EC 1.8.99.3) catalyze the reduction of sulfite to sulfide, an essential step in the anaerobic sulfate-respiration pathway. Consequently, this enzyme has been found in all dissimilatory sulfate-reducing prokaryotes (SRPs) investigated so far. Furthermore, siroheme dissimilatory sulfite reductase-like enzymes have been detected in the hyperthermophilic archaeon Pyrobaculum islandicum capable of using sulfite as terminal electron acceptor (23), the phototrophic bacterium Allochromatium vinosum (10, 12), and the obligate chemolithotrophic species Thiobacillus denitrificans (32). In the latter two organisms the dissimilatory sulfite reductase has a proposed function in sulfide oxidation.Siroheme sulfite reductases consist of at least two different polypeptides in an ␣ 2 ␤ 2 structure. The genes encoding the two subunits are found adjacent to each other in the respective genomes (see, for example, references 3, 15, 17, 18, and 35) and probably arose from duplication of an ancestral gene (3). Comparative amino acid sequence analysis of the dissimilatory sulfite reductase genes (dsrAB) has recently been used to investigate the evolutionary history of anaerobic sulfate (sulfite) respiration (...

The eukaryotic 26S proteasome controls cellular processes by degrading specific regulatory proteins. Most proteins are targeted for degradation by a signal or degron that consists of two parts: a proteasome-binding tag, typically covalently attached polyubiquitin chains, and an unstructured region that serves as the initiation region for proteasomal proteolysis. Here we have characterized how the arrangement of the two degron parts in a protein affects degradation. We found that a substrate is degraded efficiently only when its initiation region is of a certain minimal length and is appropriately separated in space from the proteasome-binding tag. Regions that are located too close or too far from the proteasome-binding tag cannot access the proteasome and induce degradation. These spacing requirements are different for a polyubiquitin chain and a ubiquitin-like (UbL) domain. Thus, arrangement and location of the proteasome initiation region affect a protein’s fate and play a central role in selecting proteins for proteasome-mediated degradation.

DNA double-strand breaks (DSBs) are the most cytotoxic form of DNA damage, with their aberrant repair linked with carcinogenesis 1,2 . The conserved error-prone Non-Homologous End-Joining (NHEJ) pathway plays a key role in determining the effects of DSB-inducing agents used to treat cancer as well as the generation of antibody and T cell receptor diversity 2,3 . Here, we applied single-particle cryo-electron microscopy (EM) to visualize two key DNA-protein complexes formed by NHEJ factors. Ku, DNA-PKcs, LigIV-XRCC4, and XLF form a Long-range synaptic complex, in which the DNA ends are held ~115 Å apart. Two DNA end-bound Ku-DNA-PKcs subcomplexes are linked by DNA-PKcs-DNA-PKcs interactions and a LigIV-XRCC4-XLF-XRCC4-LigIV scaffold. The relative orientation of the DNA-PKcs molecules suggests a mechanism for auto-phosphorylation in trans, leading to dissociation of DNA-PKcs and transition into the Short-range synaptic complex. Within this complex, the Ku-bound DNA ends are aligned for processing and ligation by the XLF-anchored scaffold, and a single LigIV catalytic domain is stably associated with a nick between the two Ku molecules, suggesting that joining of both strands of a DSB involves both LigIV molecules.

Rad23 is an adaptor protein that binds to both ubiquitinated substrates and to the proteasome. Despite its association with the proteasome, Rad23 escapes degradation. Here we show that Rad23 remains stable because it lacks an effective initiation region where the proteasome can engage the protein and unfold it. Rad23 contains several internal, unstructured loops but these are too short to act as initiation regions. Experiments with model proteins show that internal loops must be surprisingly long to engage the proteasome and support degradation. These length requirements are not specific to Rad23 and reflect a general property of the proteasome.

The proteasome controls the concentrations of most proteins in eukaryotic cells. It recognizes its protein substrates through ubiquitin tags and initiates degradation at disordered regions within the substrate. Here we find that the proteasome has pronounced preferences for the amino acid sequence composition of the regions at which it initiates degradation. Specifically, proteins where the initiation regions have biased amino acid compositions show longer half-lives in yeast. The relationship is also observed on a genomic scale in mouse cells. These preferences affect the degradation rates of proteins in vitro, can explain the unexpected stability of natural proteins in yeast, and may affect the accumulation of toxic proteins in disease. We propose that the proteasome’s sequence preferences provide a second component to the degradation code and may fine-tune protein half-life in cells.