The bacterial secondary metabolite 2,4-diacetylphloroglucinol impairs mitochondrial function and affects calcium homeostasis in Neurospora crassa.

The bacterial secondary metabolite 2,4-diacetylphloroglucinol (DAPG) is of interest as an active ingredient of biological control strains of Pseudomonas fluorescens and as a potential lead pharmaceutical molecule because of its capacity to inhibit growth of diverse microbial and non-microbial cells. The mechanism by which this occurs is unknown and in this study the filamentous fungus Neurospora crassa was used as a model to investigate the effects of DAPG on a eukaryotic cell. Colony growth, conidial germination and cell fusion assays confirmed the inhibitory nature of DAPG towards N. crassa. A number of different fluorescent dyes and fluorescent protein reporters were used to assess the effects of DAPG treatment on mitochondrial and other cellular functions. DAPG treatment led to changes in mitochondrial morphology, and rapid loss of mitochondrial membrane potential. These effects are likely to be responsible for the toxicity of DAPG. It was also found that DAPG treatment caused extracellular calcium to be taken up by conidial germlings leading to a transient increase in cytosolic free Ca(2+) with a distinct concentration dependent Ca(2+) signature.

Histone deacetylase complexes promote trinucleotide repeat expansions.

Expansions of DNA trinucleotide repeats cause at least 17 inherited neurodegenerative diseases, such as Huntington's disease. Expansions can occur at frequencies approaching 100% in affected families and in transgenic mice, suggesting that specific cellular proteins actively promote (favor) expansions. The inference is that expansions arise due to the presence of these promoting proteins, not their absence, and that interfering with these proteins can suppress expansions. The goal of this study was to identify novel factors that promote expansions. We discovered that specific histone deacetylase complexes (HDACs) promote CTG•CAG repeat expansions in budding yeast and human cells. Mutation or inhibition of yeast Rpd3L or Hda1 suppressed up to 90% of expansions. In cultured human astrocytes, expansions were suppressed by 75% upon inhibition or knockdown of HDAC3, whereas siRNA against the histone acetyltransferases CBP/p300 stimulated expansions. Genetic and molecular analysis both indicated that HDACs act at a distance from the triplet repeat to promote expansions. Expansion assays with nuclease mutants indicated that Sae2 is one of the relevant factors regulated by Rpd3L and Hda1. The causal relationship between HDACs and expansions indicates that HDACs can promote mutagenesis at some DNA sequences. This relationship further implies that HDAC3 inhibitors being tested for relief of expansion-associated gene silencing may also suppress somatic expansions that contribute to disease progression.

New functions of Ctf18-RFC in preserving genome stability outside its role in sister chromatid cohesion.

Expansion of DNA trinucleotide repeats causes at least 15 hereditary neurological diseases, and these repeats also undergo contraction and fragility. Current models to explain this genetic instability invoke erroneous DNA repair or aberrant replication. Here we show that CAG/CTG tracts are stabilized in Saccharomyces cerevisiae by the alternative clamp loader/unloader Ctf18-Dcc1-Ctf8-RFC complex (Ctf18-RFC). Mutants in Ctf18-RFC increased all three forms of triplet repeat instability--expansions, contractions, and fragility--with effect over a wide range of allele lengths from 20-155 repeats. Ctf18-RFC predominated among the three alternative clamp loaders, with mutants in Elg1-RFC or Rad24-RFC having less effect on trinucleotide repeats. Surprisingly, chl1, scc1-73, or scc2-4 mutants defective in sister chromatid cohesion (SCC) did not increase instability, suggesting that Ctf18-RFC protects triplet repeats independently of SCC. Instead, three results suggest novel roles for Ctf18-RFC in facilitating genomic stability. First, genetic instability in mutants of Ctf18-RFC was exacerbated by simultaneous deletion of the fork stabilizer Mrc1, but suppressed by deletion of the repair protein Rad52. Second, single-cell analysis showed that mutants in Ctf18-RFC had a slowed S phase and a striking G2/M accumulation, often with an abnormal multi-budded morphology. Third, ctf18 cells exhibit increased Rad52 foci in S phase, often persisting into G2, indicative of high levels of DNA damage. The presence of a repeat tract greatly magnified the ctf18 phenotypes. Together these results indicate that Ctf18-RFC has additional important functions in preserving genome stability, besides its role in SCC, which we propose include lesion bypass by replication forks and post-replication repair.

The Pseudomonas fluorescens secondary metabolite 2,4 diacetylphloroglucinol impairs mitochondrial function in Saccharomyces cerevisiae.

Pseudomonas fluorescens strains are known to produce a wide range of secondary metabolites including phenazines, siderophores, pyoluteorin, and 2,4 diacetylphloroglucinol (DAPG). DAPG is of particular interest because of its antifungal properties and because its production is associated with inhibition of phytopathogenic fungi in natural disease-suppressive soils. This trait has been exploited to develop strains of P. fluorescens that have potential application as biocontrol agents. Although the biochemistry, genetics and regulation of DAPG production have been well-studied, relatively little is known about how DAPG inhibits fungal growth and how fungi respond to DAPG. Employing a yeast model and a combination of phenotypic assays, molecular genetics and molecular physiological probes, we established that inhibition of fungal growth is caused by impairment of mitochondrial function. The effect of DAPG on yeast is largely fungistatic but DAPG also induces the formation of petite cells. Expression of the multidrug export proteins Pdr5p and Snq2p is increased by DAPG-treatment but this appears to be a secondary effect of mitochondrial damage as no role in enhancing DAPG-tolerance was identified for either Pdr5p or Snq2p.

Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption.

The SRS2 (Suppressor of RAD Six screen mutant 2) gene encodes an ATP-dependent DNA helicase that regulates homologous recombination in Saccharomyces cerevisiae. Mutations in SRS2 result in a hyper-recombination phenotype, sensitivity to DNA damaging agents and synthetic lethality with mutations that affect DNA metabolism. Several of these phenotypes can be suppressed by inactivating genes of the RAD52 epistasis group that promote homologous recombination, implicating inappropriate recombination as the underlying cause of the mutant phenotype. Consistent with the genetic data, purified Srs2 strongly inhibits Rad51-mediated recombination reactions by disrupting the Rad51-ssDNA presynaptic filament. Srs2 interacts with Rad51 in the yeast two-hybrid assay and also in vitro. To investigate the functional relevance of the Srs2-Rad51 complex, we have generated srs2 truncation mutants that retain full ATPase and helicase activities, but differ in their ability to interact with Rad51. Importantly, the srs2 mutant proteins attenuated for Rad51 interaction are much less capable of Rad51 presynaptic filament disruption. An internal deletion in Srs2 likewise diminishes Rad51 interaction and anti-recombinase activity. We also present evidence that deleting the Srs2 C-terminus engenders a hyper-recombination phenotype. These results highlight the importance of Rad51 interaction in the anti-recombinase function of Srs2, and provide evidence that this Srs2 function can be uncoupled from its helicase activity.