Enhanced expression of the DNA damage-inducible gene DIN7 results in increased mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae.

We reported previously that the product of DIN7, a DNA damage-inducible gene of Saccharomyces cerevisiae, belongs to the XPG family of proteins, which are involved in DNA repair and replication. This family includes the S. cerevisiae protein Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Interestingly, Din7p is the only member of the XPG family which specifically functions in mitochondria. We reported previously that overexpression of DIN7 results in a mitochondrial mutator phenotype. In the present study we wished to test the hypothesis that this phenotype is dependent on the nuclease activity of Din7p. For this purpose, we constructed two alleles, din7-D78A and din7-D173A, which encode proteins in which highly conserved aspartates important for the nuclease activity of the XPG proteins have been replaced by alanines. Here, we report that overexpression of the mutant alleles, in contrast to DIN7, fails to increase the frequency of mitochondrial petite mutants or erythromycin-resistant (Er) mutants. Also, overproduction of din7-D78Ap does not result in destabilization of poly GT tracts in mitochondrial DNA (mtDNA), the phenotype observed in cells that overexpress Din7p. We also show that petite mutants induced by enhanced synthesis of wild-type Din7p exhibit gross rearrangements of mtDNA, and that this correlates with enhanced recombination within the mitochondrial cyt b gene. These results suggest that the stability of the mitochondrial genome of S. cerevisiae is modulated by the level of the nuclease Din7p.

A dominant mitochondrial mutator phenotype of Saccharomyces cerevisiae conferred by msh1 alleles altered in the sequence encoding the ATP-binding domain.

In order to improve our understanding of the role of the yeast MSH1 gene in error avoidance in mitochondrial DNA, two msh1 alleles were constructed, which encode proteins with amino acid substitutions in an ATP-binding domain that is highly conserved among MutS homologs. Here, we report that moderate overexpression of the msh1-R813W or msh1-G776D allele, in strains which also carry the wild-type MSH1 allele, slightly increases the frequency of mutations conferring resistance to erythromycin (E(r)) and elevates the frequency of alterations within a polyGT tract present in mitochondrial DNA (mtDNA). This result indicates that the mutant alleles confer a dominant mitochondrial mutator phenotype and strongly suggests that the ATP-binding domain plays a crucial role in the in vivo function of Msh1p. Interestingly, we have found that overexpression of wild-type MSH1 has opposite effects on the stability of polyGT vs. polyAT tracts present in mtDNA; excess of Msh1p slightly increases the stability of polyGT tracts, whereas the stability of polyAT tracts is dramatically decreased. We show that although overexpression of msh1-R813W or msh1-G776D also results in a marked overall increase in the frequency of alterations in polyAT tracts, the spectrum of alterations differs from that found in cells overexpressing MSH1; large deletions predominate in the latter case, while 2-bp deletions are generated in cells that overproduce the mutant msh1p. This result strongly suggests that the mutations in the ATP binding domain change the specificity of the protein with respect to the recognition of potentially mutagenic structures in mtDNA.

Bacterial ion channels and their eukaryotic homologues.

Due to the relative ease of obtaining their crystal structures, bacterial ion channels provide a unique opportunity to analyse structure and function of their eukaryotic homologues. This review describes prokaryotic channels whose structures have been determined. These channels are KcsA, a bacterial homologue of eukaryotic potassium channels, MscL, a bacterial mechanosensitive ion channel and ClC0, a prokaryotic homologue of the eukaryotic ClC family of anion-selective channels. General features of their structure and function are described with a special emphasis on the advantages that these channels offer for understanding the properties of their eukaryotic homologues. We present amino-acid sequences of eukaryotic proteins related in their primary sequences to bacterial mechanosensitive channels. The usefulness of bacterial mechanosensitive channels for the studies on general principles of mechanosensation is discussed.

[Bacterial ion canals]. / Bakteryjne kanaly jonowe.

After an overview of potassium and chloride ion channels found in bacterial inner membrane this review focuses on mechanosensitive ion channels from the inner membrane of Escherichia coli. Mechanosensitive channels, MscL and MscS, have major roles in managing the transition from high to low environments. Biochemical and genetic studies of MscL, combined with structural information derived from X-ray crystalography, have brought the knowledge of how a mechanosensitive channel senses membrane tension. Physiological studies on MscL and on MscS have demonstrated how the mechanosensitive channels contribute to the bacterial membrane response upon hypo-osmotic stress.

The product of the DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, specifically functions in mitochondria.

We reported previously that the product of the DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, belongs to a family of proteins that are involved in DNA repair and replication. The family includes S. cerevisiae proteins Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Here, we report that Din7p specifically affects metabolism of mitochondrial DNA (mtDNA). We have found that dun1 strains, defective in the transcriptional activation of the DNA damage-inducible genes RNR1, RNR2, and RNR3, exhibit an increased frequency in the formation of the mitochondrial petite (rho(-)) mutants. This high frequency of petites arising in the dun1 strains is significantly reduced by the din7::URA3 allele. On the other hand, overproduction of Din7p from the DIN7 gene placed under control of the GAL1 promoter dramatically increases the frequency of petite formation and the frequency of mitochondrial mutations conferring resistance to erythromycin (E(r)). The frequencies of chromosomal mutations conferring resistance to canavanine (Can(r)) or adenine prototrophy (Ade(+)) are not affected by enhanced synthesis of Din7p. Experiments using Din7p fused to the green fluorescent protein (GFP) and cell fractionation experiments indicate that the protein is located in mitochondria. A possible mechanism that may be responsible for the decreased stability of the mitochondrial genome in S. cerevisiae cells with elevated levels of Din7p is discussed.

Glutathione (GSH) reduces the open probability of mechanosensitive channels in Escherichia coli protoplasts.

The effects of glutathione (reduced GSH, and oxidized GSSG) on mechanosensitive (MS) ion channels from Escherichia coli protoplasts were investigated using excised, inside-out membrane patches. Our studies demonstrate here that 5 mM GSH irreversibly reduces the activities of the 560-pS MS channel by approximately 70-75% whereas 5 mM GSSG did not alter the MS channel currents. In addition, millimolar concentrations of dithiothreitol had similar although weaker effects to GSH. The physiological concentration of GSH in E. coli cytoplasm ranges from 3.5 mM to 6.6 mM, which may indicate that under normal conditions these MS channels open less due to membrane stretch.

Voltage-independent adaptation of mechanosensitive channels in Escherichia coli protoplasts.

Mechanosensitive (MS) ion channels, with 560 pS conductance, opened transiently by rapid application of suction pulses to patches of E. coli protoplast membrane. The adaptation phase of the response was voltage-independent. Application of strong suction pulses, which were sufficient to cause saturation of the MS current, did not abolish the adaptation. Multiple-pulse experimental protocols revealed that once MS channels had fully adapted, they could be reactivated by a second suction pulse of similar amplitude, providing the time between pulses was long enough and suction had been released between pulses. Limited proteolysis (0.2 mg/ml pronase applied to the cytoplasmic side of the membrane patch) reduced the number of open channels without affecting the adaptation. Exposing patches to higher levels of pronase (1 mg/ml) removed responsiveness of the channel to suction and abolished adaptation consistent with disruption of the tension transmission mechanism responsible for activating the MS channel. Based on these data we discuss a mechanism for mechanosensitivity mediated by a cytoplasmic domain of the MS channel molecule or associated protein.

Interstitial cystitis: successful management by increasing urinary voiding intervals.

A variant of the interstitial cystitis (IC) syndrome, minimal or no pain or significant pain that has been lessened by another therapy, can be clinically improved by retraining of the individual's voiding pattern. Patients with greater pain but capable of completing this protocol also may be helped. While this group of patients fills the diagnostic criteria of IC and has many of the classic changes, many differ in that they have minimal or no pain, while simultaneously they have a dysfunctional bladder as expected from long-term low-volume voiding. All patients were placed on a protocol which focused on progressively increasing intervals between voids. Fifteen to thirty minutes initially were added to their present voiding time. The same increase was added to the voiding pattern every three or four weeks until an interval of three to four hours between voids was achieved. Twenty-one patients fit the criteria to be entered into this study. Overall 71 percent (15/21) had successful management of their symptoms and reported a 50 percent decrease in their symptoms of urinary urgency, frequency, and nocturia. Nineteen percent (4/21) reported 25 percent decrease in symptoms and 10 percent had no change. Presence of significant pain adversely affects outcome, 3/7 (with pain) improvements versus 12/14 (without pain). For all patients there was a significant increase in bladder capacity (92 mL average before study and 179 mL after) and average daily voids (13.2/day prestudy and 7.4 post-therapy). These differences in voided profiles were statistically significant (p value less than 0.01).

The chronic patient with a nonpsychotic diagnosis.

In a population of almost 1,100 patients in an urban hospital's aftercare program for the chronically mentally ill, 137 patients (12.5 per cent) were found to have nonpsychotic diagnoses. Patients with nonpsychotic diagnoses tended to be older at the first psychiatric contact, a preponderance were first-born or only children, and a higher percentage had been or were presently married. The therapist's possible role in the prevention of chronicity in nonpsychotic patients is discussed; therapists may contribute to the creation of chronic patients, the authors say, by fostering the emergence of increased dependency needs in patients who initially present with acute symptoms. The limitations of psychotherapeutic intervention must be stressed to psychotherapy students, and psychotherapy with nonpsychotic patients should be time-limited and focused on problem-solving.