Potentiation of oxygen toxicity by menadione in Saccharomyces cerevisiae

The cytotoxicity of molecular oxygen can be sharply increased in the yeast Saccharomyces cerevisiae by the use of redox compounds capable of shunting electrons in vivo and of spontaneous reoxidation under aerobic conditions. Among these redox compounds, menadione (Vitamin K3) is particularly able to stimulate the cyanide-resistant respiration of the yeast cells. Under steady-state conditions, the efficiency of menadione is modulated by the physiological state of the yeast cells and also depends on the availability of reducing agents within the cell. Menadione shows lethal effects towards yeast cells in the presence of O2 only, as a result of the production of toxic metabolites like O2-. and H2O2 which are actually detected in the extracellular fluid. Inhibitors of the enzymes scavenging O2-. and H2O2 generally potentiate the lethal effects of this redox compound. On the other hand, superoxide dismutase and/or catalase supplemented into the incubation buffer have been found to protect the cells to various extents from the cytotoxic effects of menadione. Our data support the following conclusions: When the cellular enzymatic defences are functional, the moderate lethality induced by menadione is principally mediated by O2-. ions acting on the outer side of the cell (peripheral region). In the presence of cyanide, but not of azide, the loss of viability also results from additional damage occurring within the inner cell region. In this case, intracellular injury can be caused by H2O2 alone but our data also suggest that during redox cycling more reactive species--O2-. and probably OH.--are generally intracellularly and are involved in the cytotoxic process.     

Saccharomyces cerevisiae RAP1 binds to telomeric sequences with spatial flexibility

A wide divergence has been detected in the telomeric sequences among budding yeast species. Despite their length and homogeneity differences, all these yeast telomeric sequences show a conserved core which closely matches the consensus RAP1-binding sequence. We demonstrate that the RAP1 protein binds this sequence core, without involving the diverged sequences outside the core. In Saccharomyces castellii and Saccharomyces dairensis specific classes of interspersed variant repeats are present. We show here that a RAP1-binding site is formed in these species by connecting two consecutive 8 bp telomeric repeats. DNase I footprint analyses specify the binding site as the 13 bp sequence CTGGGTGTCTGGG. The RAP1 protein also binds the variant repeats, although with a lowered affinity. However, a split footprint is produced when RAP1 binds a variant repeat where the two half-sites of the binding site are separated by an additional 6 nt. This is probably caused by the intervening sequence looping out of the RAP1-DNA complex. We suggest that the bipartite subdomain structure of the RAP1 protein allows it to remodel telomeric chromatin, a feature which may be of great relevance for telomeric chromatin assembly and structure in vivo.     

Potentiation of gene targeting in human cells by expression of Saccharomyces cerevisiae Rad52

When exogenous DNA is stably introduced in mammalian cells, it is typically integrated in random positions, and only a minor fraction enters a pathway of homologous recombination (HR). The complex Rad51/Rad52 is a major player in the management of exogenous DNA in eukaryotic organisms and plays a critical role in the choice of repair system. In Saccharomyces cerevisiae, the pathway of choice is HR, mediated by Rad52 (ScRad52), which differs slightly from its human homologue. Here, we present an approach that utilizes ScRad52 to enhance HR in human cells containing a specific substrate for recombination. Clones of HeLa cells were produced expressing functional ScRad52. These cells showed enhanced resistance to DNA damaging treatments and revealed a different distribution of Rad51 foci (a marker of recombination complex formation). More significantly, ScRad52 expression resulted in an up to 37-fold increase in gene targeting by HR. In the same cells, random integration of exogenous DNA was significantly reduced, consistent with the view that HR and non-homologous end joining are alternative competing pathways. Expression of ScRad52 could offer a major improvement for experiments requiring gene targeting by HR, both in basic research and in gene therapy studies.     

Chromatin structures of Kluyveromyces lactis centromeres in K. lactis and Saccharomyces cerevisiae

We have investigated the chromatin structure of Kluyveromyces lactis centromeres in isolated nuclei of K. lactis and Saccharomyces cerevisiae by using micrococcal nuclease and DNAse I digestion. The protected region found in K. lactis is approximately 270 bp long and encompasses the centromeric DNA elements, KlCDEI, KlCDEII, and KlCDEIII, but not KlCDE0. Halving KlCDEII to 82 bp impaired centromere function and led to a smaller protected structure (210 bp). Likewise, deletion of 5 bp from KlCDEI plus adjacent flanking sequences resulted in a smaller protected region and a decrease in centromere function. The chromatin structures of KlCEN2 and KlCEN4 present on plasmids were found to be similar to the structures of the corresponding centromeres in their chromosomal context. A different protection pattern of KlCEN2 was detected in S. cerevisiae, suggesting that KlCEN2 is not properly recognized by at least one of the centromere binding proteins of S. cerevisiae. The difference is mainly found at the KlCDEIII side of the structure. This suggests that one of the components of the ScCBF3-complex is not able to bind to KlCDEIII, which could explain the species specificity of K. lactis and S. cerevisiae centromeres.     

C-terminal truncation of RAP1 results in the deregulation of telomere size, stability, and function in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae DNA-binding protein RAP1 is capable of binding in vitro to sequences from a wide variety of genomic loci, including upstream activating sequence elements, the HML and HMR silencer regions, and the poly(G1-3T) tracts of telomeres. Recent biochemical and genetic studies have suggested that RAP1 physically and functionally interacts with the yeast telomere. To further investigate the role of RAP1 at the telomere, we have identified and characterized three intragenic suppressors of a temperature-sensitive allele of RAP1, rap1-5. These telomere deficiency (rap1t) alleles confer several novel phenotypes. First, telomere tract size elongates to up to 4 kb greater than sizes of wild-type or rap1-5 telomeres. Second, telomeres are highly unstable and are subject to rapid, but reversible, deletion of part or all of the increase in telomeric tract length. Telomeric deletion does not require the RAD52 or RAD1 gene product. Third, chromosome loss and nondisjunction rates are elevated 15- to 30-fold above wild-type levels. Sequencing analysis has shown that each rap1t allele contains a nonsense mutation within a discrete region between amino acids 663 and 684. Mobility shift and Western immunoblot analyses indicate that each allele produces a truncated RAP1 protein, lacking the C-terminal 144 to 165 amino acids but capable of efficient DNA binding. These data suggest that RAP1 is a central regulator of both telomere and chromosome stability and define a C-terminal domain that, while dispensable for viability, is required for these telomeric functions.     

Chromatin remodelling complex RSC promotes base excision repair in chromatin of Saccharomyces cerevisiae

The base excision repair (BER) pathway is a conserved DNA repair system required to maintain genomic integrity and prevent mutagenesis in all eukaryotic cells. Nevertheless, how BER operates in vivo (i.e. in the context of chromatin) is poorly understood. We have investigated the role of an essential ATP-dependent chromatin remodelling (ACR) complex RSC (Remodels the Structure of Chromatin) in BER of intact yeast cells. We show that depletion of STH1, the ATPase subunit of RSC, causes enhanced sensitivity to the DNA alkylating agent methyl methanesulfonate (MMS) and results in a substantial inhibition of BER, at the GAL1 locus and in the genome overall. Consistent with this observation, the DNA in chromatin is less accessible to micrococcal nuclease digestion in the absence of RSC. Quantitative PCR results indicate that repair deficiency in STH1 depleted cells is not due to changes in the expression of BER genes. Collectively, our data indicates the RSC complex promotes efficient BER in chromatin. These results provide, for the first time, a link between ATP-dependent chromatin remodelling and BER in living cells.