The translesion synthesis (TLS) polymerases Polζ and Rev1 form a complex that enables replication of damaged DNA. The Rev7 subunit of Polζ, which is a multifaceted HORMA (Hop1, Rev7, Mad2) protein with roles in TLS, DNA repair, and cell-cycle control, facilitates assembly of this complex by binding Rev1 and the catalytic subunit of Polζ, Rev3. Rev7 interacts with Rev3 by a mechanism conserved among HORMA proteins, whereby an open-to-closed transition locks the ligand underneath the "safety belt" loop. Dimerization of HORMA proteins promotes binding and release of this ligand, as exemplified by the Rev7 homolog, Mad2. Here, we investigate the dimerization of Rev7 when bound to the two Rev7-binding motifs (RBMs) in Rev3 by combining in vitro analyses of Rev7 structure and interactions with a functional assay in a Rev7 cell line. We demonstrate that Rev7 uses the conventional HORMA dimerization interface both to form a homodimer when tethered by the two RBMs in Rev3 and to heterodimerize with other HORMA domains, Mad2 and p31 Structurally, the Rev7 dimer can bind only one copy of Rev1, revealing an unexpected Rev1/Polζ architecture. In cells, mutation of the Rev7 dimer interface increases sensitivity to DNA damage. These results provide insights into the structure of the Rev1/Polζ TLS assembly and highlight the function of Rev7 homo- and heterodimerization.

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eThe Gram-positive, anaerobic, spore-forming bacterium Clostridium perfringens causes a variety of diseases in both humans and animals, and spore germination is thought to be the first stage of C. perfringens infection. Previous studies have indicated that the germinant receptor (GR) proteins encoded by the bicistronic gerKA-gerKC operon as well as the proteins encoded by the gerKB and gerAA genes are required for normal germination of C. perfringens spores. We now report the individual role of these GR proteins by analyzing the germination of strains carrying mutations in gerKA, gerKC, or both gerKB and gerAA. Western blot analysis was also used to determine the location and numbers of GerKC proteins in spores. Conclusions from this work include the following: (i) gerKC mutant spores germinate extremely poorly with KCl, L-asparagine, a mixture of asparagine and KCl, or NaP i ; (ii) gerKC spores germinate significantly more slowly than wild-type and other GR mutant spores with a 1:1 chelate of Ca 2؉ and dipicolinic acid and very slightly more slowly with dodecylamine; (iii) the germination defects in gerKC spores are largely restored by expressing the wild-type gerKA-gerKC operon in trans; (iv) GerKC is required for the spores' viability, almost certainly because of the gerKC spores' poor germination; and (v) GerKC is located in the spores' inner membrane, with ϳ250 molecules/spore. Collectively, these results indicate that GerKC is the main GR protein required for nutrient and nonnutrient germination of spores of C. perfringens food-poisoning isolates.

The F-box proteins are the substrate recognition subunits of the SCF (Skp1-Cul1-Rbx1-F-box protein) ubiquitin ligase complexes that control the stability of numerous regulators in eukaryotic cells. Here we show that dimerization of the F-box protein Fbx4 is essential for SCF Fbx4 (the superscript denotes the F-box protein) ubiquitination activity toward the telomere regulator Pin2 (also known as TRF1). The crystal structure of Ubiquitin-mediated proteolysis governs the levels of many cellular proteins via a triple-enzyme cascade, involving ubiquitin-activating (E1), -conjugating (E2), and -ligating (E3) activities (1, 2). The E3 ubiquitin ligases recruit both ubiquitincharged E2 and the targeted substrate so as to confer substrate specificity and mediate ubiquitin transfer from E2 to the substrate (2). RING-based E3s, one of the two major classes of E3s, likely use a zinc-binding RING structural motif to recruit E2 and thus facilitate direct transfer of ubiquitin from E2 onto its substrate (2). The most intensively studied RING E3s are members of the cullin-RING ligase superfamily, which is now recognized as the largest known class of E3 ligases (3-7).

The nutrient germinant receptors (nGRs) of spores of Bacillus species are clusters of three proteins that play a critical role in triggering the germination of dormant spores in response to specific nutrient molecules. Here we report the crystal structure of the C protein of the GerB germinant receptor, so called GerBC, of Bacillus subtilis spores at 2.3 Å resolution. The GerBC protein adopts a previously uncharacterized type of protein fold consisting of three distinct domains, each of which is centered by a β sheet surrounded by multiple α helices. Secondary structure prediction and structure-based sequence alignment suggest that the GerBC structure represents the prototype for C subunits of nGRs from spores of all Bacillales and Clostridiales species and defines two highly conserved structural regions in this family of proteins. GerBC forms an interlocked dimer in the crystalline state but is predominantly monomeric in solution, pointing to the possibility that GerBC oligomerizes as a result of either high local protein concentrations or interaction with other nGR proteins in spores. Our findings provide the first structural view of the nGR subunits and a molecular framework for understanding the architecture, conservation and function of nGRs.

Highly conserved amino acid residues in the C subunits of the germinant receptors (GRs) of spores of Bacillus and Clostridium species have been identified by amino acid sequence comparisons, as well as structural predictions based on the high-resolution structure recently determined for the C subunit of the Bacillus subtilis GerB GR (GerBC). Single and multiple alanine substitutions were made in these conserved residues in three regions of GerBC, and the effects of these changes on B. subtilis spore germination via the GerB GR alone or in concert with the GerK GR, as well as on germination via the GerA GR, were determined. In addition, levels of the GerBC variants in the spore inner membrane were measured, and a number of the GerBC proteins were expressed and purified and their solubility and aggregation status were assessed. This work has done the following: (i) identified a number of conserved amino acids that are crucial for GerBC function in spore germination via the GerB GR and that do not alter spores' levels of these GerBC variants; (ii) identified other conserved GerBC amino acid essential for the proper folding of the protein and/or for assembly of GerBC in the spore inner membrane; (iii) shown that some alanine substitutions in GerBC significantly decrease the GerA GR's responsiveness to its germinant L-valine, consistent with there being some type of interaction between GerA and GerB GR subunits in spores; and (iv) found no alanine substitutions that specifically affect interaction between the GerB and GerK GRs.The ability to form spores is a remarkable property shared by many Bacillus species (23). Spores of these bacteria are formed in sporulation, a process triggered by starvation for one or more nutrients. These spores are metabolically dormant and extremely resistant to all manner of environmental stress factors (22, 23) and can remain in this dormant, resistant state for years. However, when conditions are favorable for growth, spores can rapidly return to life in the process of germination followed by outgrowth (11,19,21). A major signal that promotes germination is the presence of specific nutrients called germinants in the spore's environment. These nutrients are sensed by germinant receptors (GRs) located in the spore's inner membrane, and it is very likely that the binding of a nutrient germinant with its cognate GR triggers subsequent germination events.Bacillus subtilis spores contain three major GRs, termed GerA, GerB, and GerK (22, 23). The GerA GR responds to L-alanine or L-valine, while the GerB and GerK GRs cooperate to respond to a mixture of L-asparagine, D-glucose, D-fructose, and K ϩ ions (termed AGFK). Each of these GRs is encoded by a tricistronic operon, and the three polypeptides encoded by each operon (termed A, B, and C) are essential for specific GR activities and most likely form a multisubunit complex. The A and B subunits of each GR are likely to be polytopic integral membrane proteins, while the C subunit is a peripheral membrane protein held in the membrane by a diacy...

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