Sumoylation during genotoxic stress regulates the composition of DNA repair complexes. The yeast metalloprotease Wss1 clears chromatin-bound sumoylated proteins. Wss1 and its mammalian analog, DVC1/Spartan, belong to minigluzincins family of proteases. Wss1 proteolytic activity is regulated by a cysteine switch mechanism activated by chemical stress and/or DNA binding. Wss1 is required for cell survival following UV irradiation, the smt3-331 mutation and Camptothecin-induced formation of covalent topoisomerase 1 complexes (Top1cc). Wss1 forms a SUMO-specific ternary complex with the AAA ATPase Cdc48 and an adaptor, Doa1. Upon DNA damage Wss1/Cdc48/Doa1 is recruited to sumoylated targets and catalyzes SUMO chain extension through a newly recognized SUMO ligase activity. Activation of Wss1 results in metalloprotease self-cleavage and proteolysis of associated proteins. In cells lacking Tdp1, clearance of topoisomerase covalent complexes becomes SUMO and Wss1-dependent. Upon genotoxic stress, Wss1 is vacuolar, suggesting a link between genotoxic stress and autophagy involving the Doa1 adapter.DOI: http://dx.doi.org/10.7554/eLife.06763.001

Abstract. Dictyostelium discoideum initiates development when ceils overgrow their bacterial food source and starve. To coordinate development, the cells monitor the extracellular level of a protein, conditioned medium factor (CMF), secreted by starved cells. When a majority of the cells in a given area have starved, as signaled by CMF secretion, the extracellular level of CMF rises above a threshold value and permits aggregation of the starved cells. The cells aggregate using relayed pulses of cAMP as the chemoattractant. Cells in which CMF accumulation has been blocked by antisense do not aggregate except in the presence of exogenous CMF. We find that these cells are viable but do not chemotax towards cAMP. Videomicroscopy indicates that the inability of CMF antisense cells to chemotax is not due to a gross defect in motility, although both video and scanning electron microscopy indicate that CMF increases the frequency of pseudopod formation.The activations of Ca 2+ influx, adenylyl cyclase, and guanylyl cyclase in response to a pulse of cAMP are strongly inhibited in cells lacking CMF, but are rescued by as little as 10 s exposure of cells to CMF. The activation of phospholipase C by cAMP is not affected by CMF. Northern blots indicate normal levels of the cAMP receptor mRNA in CMF antisense cells during development, while cAMP binding assays and Scatchard plots indicate that CMF antisense cells contain normal levels of the cAMP receptor. In Dictyostelium, both adenylyl and guanylyl cyclases are activated via G proteins. We find that the interaction of the cAMP receptor with G proteins in vitro is not measurably affected by CMF, whereas the activation of adenylyl cyclase by G proteins requires cells to have been exposed to CMF. CMF thus appears to regulate aggregation by regulating an early step of cAMP signal transduction.

When the unicellular eukaryote Dictyostelium discoideum starves, it senses the local density of other starving cells by simultaneously secreting and sensing a glycoprotein called conditioned medium factor (CMF). When the density of starving cells is high, the corresponding high density of CMF permits signal transduction through cAR1, the chemoattractant cAMP receptor. cAR1 activates a heterotrimeric G protein whose ␣-subunit is G␣2. CMF regulates cAMP signal transduction in part by regulating the lifetime of the cAMP-stimulated G␣2-GTP configuration. We find here that guanosine 5-3-O-(thio)triphosphate (GTP␥S) inhibits the binding of CMF to membranes, suggesting that the putative CMF receptor is coupled to a G protein. Cells lacking G␣1 (G␣1 null) do not exhibit GTP␥S inhibition of CMF binding and do not exhibit CMF regulation of cAMP signal transduction, suggesting that the putative CMF receptor interacts with G␣1. Work by others has suggested that G␣1 inhibits phospholipase C (PLC), yet when cells lacking either G␣1 or PLC were starved at high cell densities (and thus in the presence of CMF), they developed normally and had normal cAMP signal transduction. We find that CMF activates PLC. G␣1 null cells starved in the absence or presence of CMF behave in a manner similar to control cells starved in the presence of CMF in that they extend pseudopods, have an activated PLC, have a low cAMP-stimulated GTPase, permit cAMP signal transduction, and aggregate. Cells lacking G␤ have a low PLC activity that cannot be stimulated by CMF. Cells lacking PLC exhibit IP 3 levels and cAMPstimulated GTP hydrolysis rates intermediate to what is observed in wild-type cells starved in the absence or in the presence of an optimal amount of CMF. We hypothesize that CMF binds to its receptor, releasing G␤␥ from G␣1. This activates PLC, which causes the G␣2 GTPase to be inhibited, prolonging the lifetime of the cAMPactivated G␣2-GTP configuration. This, in turn, allows cAR1-mediated cAMP signal transduction to take place.

The argU (dnaY) gene of Escherichia coli is located, in clockwise orientation, at 577.5 kilobases (kb) on the chromosome physical map. There was a cryptic prophage spanning the 2 kb immediately downstream of argU that consisted of sequences similar to the phage P22 int gene, a portion of the P22 xis gene, and portions of the exo, P, and ren genes of bacteriophage X. This cryptic prophage was designated DLP12, for defective lambdoid prophage at 12 min. Immediately clockwise of DLP12 was the IS3 a4,(4 insertion element. The argU and DLP12int genes overlapped at their 3' ends, and argU contained sequence homologous to a portion of the phage P22 attP site. Additional homologies to lambdoid phages were found in the 25 kb clockwise of argU. These included the cryptic prophage qsr' (P. J. Highton, Y. Chang, W. R. Marcotte, Jr., and C. A. Schnaitman, J. Bacteriol. 162:256-262, 1985), a sequence homologous to a portion of X orf-194, and an attR homolog. Inasmuch as the DLP12 att int xis exo Piren region, the qsr' region, and homologs of orf-194 and attR were arranged in the same order and orientation as the lambdoid prophage counterparts, we propose that the designation DLP12 be applied to all these sequences. This organization of the DLP12 sequences and the presence of the argU/DLP12 int pair in several E. coli strains and closely related species suggest that DLP12 might be an ancestral lambdoid prophage. Moreover, the presence of similar sequences at the junctions of DLP12 segments and their phage counterparts suggests that a common mechanism could have transferred these DLP12 segments to more recent phages.The Escherichia coli argU gene encodes a minor arginine tRNA, tRNAUg , which corresponds to the rare codon AGA and possibly AGG (18,39,51). (This gene was originally called dna Y because a temperature-sensitive mutation caused preferential inhibition of DNA synthesis at high temperature [24]; it is now redesignated argU after the convention of Fournier and Ozeki [17].) argU expression has several unusual characteristics. First, it is transcribed into a monocistronic message, whereas most tRNA genes are parts of operons (17). Second, transcription in vitro is terminated by a rho-dependent process to generate 180-and 190-nucleotide products. The precursors are presumably processed at the 5' and 3' ends to generate the 77-nucleotide mature form.Third, tRNAUAg is a minor species, suggesting inefficient transcription. However, the argU promoter should be strong, based on its sequence and spacing (23). This suggests negative control of argU, and a potential regulator site of 31 base pairs (bp) of dyad symmetry is located downstream of the promoter -10 sequence. Fourth, a 387-codon open reading frame (ORF) extends toward and overlaps the 3' end of argU (51). This ORF is especially interesting because it contains the extremely high number of 13 AGA and AGG codons-the argU tRNA cognates. Understanding argU regulation and the relationship of the downstream ORF might provide clues about the role of argU in replication, which ...