N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation in a RecA strain of Escherichia coli

274 N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced forward mutations in the lacI gene of an Escherichia coli RecA- strain were cloned and sequenced. Base substitutions accounted for 264 mutations and consisted of 261 G:C----A:T transitions (including one double mutant with two G:C----A:T transitions separated by 25 base pairs), two A:T----G:C transitions and one A:T----T:A transversion. Therefore, 263 of the 274 mutations (all the transitions) can be explained as a result of the direct mispairing of O6-methylguanine, and O4-methylthymine residues during DNA synthesis. The source of the transversion is not known. The remaining mutations, one 16-base pair deletion, two -1 frameshifts and 7 frameshifts at the lacI frameshift hotspot, are located in runs of identical bases or flanked by directly repeated DNA sequences and can therefore be explained by template slippage events during DNA synthesis. The observed distribution of mutations recovered is identical to that found in a RecA+ background indicating little involvement of RecA function in MNNG-induced mutation. Analysis of neighbouring base sequence revealed that the G:C----A:T transition was 6 times more likely to be recovered if the mutated guanine residue was preceded by a purine rather than a pyrimidine. A most striking aspect of this distribution concerns particular residues in the core domain of the lac repressor protein. Within this domain the great majority of mutations generate nonsense codons or alter Gly codons.     

Mutagenesis resulting from depurination is an SOS process

When bacteriophage phi X174 am3 DNA depurinated in vitro is transfected into E. coli spheroplasts prepared from bacteria previously exposed to UV light, a strong mutagenic response is observed. This mutagenic response does not occur in spheroplasts derived from pre-irradiated bacteria carrying defective recA, recF or umuC genes. These findings indicate that mutagenesis at apurinic sites is an SOS-dependent process. The mutagenic response is not dependent on the multiplicity of transfection. This suggests that mutagenesis is not mediated by recombination.     

Screening for mevalonate biosynthetic pathway inhibitors using sensitized bacterial strains

A simple, optical density-based assay for inhibitors of the mevalonate-dependent pathway for isoprenoid biosynthesis was developed. The assay uses pathway-sensitized Staphylococcus aureus strains and is fully compatible with high-density screening in a 1536-well format. S. aureus strains were constructed in which genes required for mevalonate-dependent isopentenyl pyrophosphate (IPP) synthesis were regulated by an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible promoter. Inhibitors of the target enzymes displayed greater antibacterial potency in media containing low concentrations of IPTG, and therefore less induction of mevalonate pathway genes, than in media with high IPTG conditions. This differential growth phenotype was exploited to bias the cell-based screening hits toward specific inhibitors of mevalonate-dependent IPP biosynthesis. Screens were run against strains engineered for regulation of the enzymes HMG-CoA synthase (MvaS) and mevalonate kinase (mvaK1), mevalonate diphosphate decarboxylase (mvaD), and phosphomevalonate kinase (mvaK2). The latter three enzymes are regulated as an operon. These assays resulted in the discovery of potent antibacterial hits that were progressed to an active hit-to-lead program. The example presented here demonstrates that a cell sensitization strategy can be successfully applied to a 1.3-million compound high-throughput screen in a high-density 1536-well format.     

Proteasome activator 200: the heat is on..

Proteasomes play a key regulatory role in all eukaryotic cells by removing proteins in a timely manner. There are two predominant forms: The 20S core particle (CP) can hydrolyze peptides and certain unstructured proteins, and the 26S holoenzyme is able to proteolyse most proteins conjugated to ubiquitin. The 26S complex consists of a CP barrel with a 19S regulatory particle (RP; a.k.a PA700) attached to its outer surface. Several studies purified another proteasome activator with a MW of 200 kDa (PA200) that attaches to the same outer ring of the CP. A role for PA200 has been demonstrated in spermatogenesis, in response to DNA repair and in maintenance of mitochondrial inheritance. Enhanced levels of PA200-CP complexes are observed under conditions in which either activated or disrupted CP prevail, suggesting it participates in regulating overall proteolytic activity. PA200, or its yeast ortholog Blm10, may also incorporate into 26S proteasomes yielding PA200-CP-RP hybrids. A three-dimensional molecular structure determined by x-ray crystallography of Blm10-CP provides a model for activation. The carboxy terminus of Blm10 inserts into a dedicated pocket in the outer ring of the CP surface, whereas multiple HEAT-like repeats fold into an asymmetric solenoid wrapping around the central pore to stabilize a partially open conformation. The resulting hollow domelike structure caps the entire CP surface. This asymmetric structure may provide insight as to how the 19S RP, with two HEAT repeatlike subunits (Rpn1, Rpn2) alongside six ATPases (Rpt1-6), attaches to the same surface of the CP ring, and likewise, induces pore opening.     

A gamma interferon independent mechanism of CD4 T cell mediated control of M. tuberculosis infection in vivo

CD4 T cell deficiency or defective IFNγ signaling render humans and mice highly susceptible to Mycobacterium tuberculosis (Mtb) infection. The prevailing model is that Th1 CD4 T cells produce IFNγ to activate bactericidal effector mechanisms of infected macrophages. Here we test this model by directly interrogating the effector functions of Th1 CD4 T cells required to control Mtb in vivo. While Th1 CD4 T cells specific for the Mtb antigen ESAT-6 restrict in vivo Mtb growth, this inhibition is independent of IFNγ or TNF and does not require the perforin or FAS effector pathways. Adoptive transfer of Th17 CD4 T cells specific for ESAT-6 partially inhibited Mtb growth while Th2 CD4 T cells were largely ineffective. These results imply a previously unrecognized IFNγ/TNF independent pathway that efficiently controls Mtb and suggest that optimization of this alternative effector function may provide new therapeutic avenues to combat Mtb through vaccination.     

Blm10 facilitates nuclear import of proteasome core particles

Short‐lived proteins are degraded by proteasome complexes, which contain a proteolytic core particle (CP) but differ in the number of regulatory particles (RPs) and activators. A recently described member of conserved proteasome activators is Blm10. Blm10 contains 32 HEAT‐like modules and is structurally related to the nuclear import receptor importin/karyopherin β. In proliferating yeast, RP‐CP assemblies are primarily nuclear and promote cell division. During quiescence, RP‐CP assemblies dissociate and CP and RP are sequestered into motile cytosolic proteasome storage granuli (PSG). Here, we show that CP sequestration into PSG depends on Blm10, whereas RP sequestration into PSG is independent of Blm10. PSG rapidly clear upon the resumption of cell proliferation and proteasomes are relocated into the nucleus. Thereby, Blm10 facilitates nuclear import of CP. Blm10‐bound CP serves as an import receptor–cargo complex, as Blm10 mediates the interaction with FG‐rich nucleoporins and is dissociated from the CP by Ran‐GTP. Thus, Blm10 represents the first CP‐dedicated nuclear import receptor in yeast.     

RecO Protein Initiates DNA Recombination and Strand Annealing through Two Alternative DNA Binding Mechanisms

Recombination mediator proteins (RMPs) are important for genome stability in all organisms. Several RMPs support two alternative reactions: initiation of homologous recombination and DNA annealing. We examined mechanisms of RMPs in both reactions with Mycobacterium smegmatis RecO (MsRecO) and demonstrated that MsRecO interacts with ssDNA by two distinct mechanisms. Zinc stimulates MsRecO binding to ssDNA during annealing, whereas the recombination function is zinc-independent and is regulated by interaction with MsRecR. Thus, different structural motifs or conformations of MsRecO are responsible for interaction with ssDNA during annealing and recombination. Neither annealing nor recombinase loading depends on MsRecO interaction with the conserved C-terminal tail of single-stranded (ss) DNA-binding protein (SSB), which is known to bind Escherichia coli RecO. However, similarly to E. coli proteins, MsRecO and MsRecOR do not dismiss SSB from ssDNA, suggesting that RMPs form a complex with SSB-ssDNA even in the absence of binding to the major protein interaction motif. We propose that alternative conformations of such complexes define the mechanism by which RMPs initiate the repair of stalled replication and support two different functions during recombinational repair of DNA breaks.     

Converting cancer therapies into cures: lessons from infectious diseases

During the past decade, cancer drug development has shifted from a focus on cytotoxic chemotherapies to drugs that target specific molecular alterations in tumors. Although these drugs dramatically shrink tumors, the responses are temporary. Research is now focused on overcoming drug resistance, a frequent cause of treatment failure. Here we reflect on analogous challenges faced by researchers in infectious diseases. We compare and contrast the resistance mechanisms arising in cancer and infectious diseases and discuss how approaches for overcoming viral and bacterial infections, such as HIV and tuberculosis, are instructive for developing a more rational approach for cancer therapy. In particular, maximizing the effect of the initial treatment response, which often requires synergistic combination therapy, is foremost among these approaches. A remaining challenge in both fields is identifying drugs that eliminate drug-tolerant "persister" cells (infectious disease) or tumor-initiating/stem cells (cancer) to prevent late relapse and shorten treatment duration.     

Yeast antizyme mediates degradation of yeast ornithine decarboxylase by yeast but not by mammalian proteasome: new insights on yeast antizyme

Mammalian antizyme (mAz) is a central element of a feedback circuit regulating cellular polyamines by accelerating ornithine decarboxylase (ODC) degradation and inhibiting polyamine uptake. Although yeast antizyme (yAz) stimulates the degradation of yeast ODC (yODC), we show here that it has only a minor effect on polyamine uptake by yeast cells. A segment of yODC that parallels the Az binding segment of mammalian ODC (mODC) is required for its binding to yAz. Although demonstrating minimal homology to mAz, our results suggest that yAz stimulates yODC degradation via a similar mechanism of action. We demonstrate that interaction with yAz provokes degradation of yODC by yeast but not by mammalian proteasomes. This differential recognition may serve as a tool for investigating proteasome functions.