Telomerase Deficiency Affects the Formation of Chromosomal Translocations by Homologous Recombination in Saccharomyces cerevisiae

Telomerase is a ribonucleoprotein complex required for the replication and protection of telomeric DNA in eukaryotes. Cells lacking telomerase undergo a progressive loss of telomeric DNA that results in loss of viability and a concomitant increase in genome instability. We have used budding yeast to investigate the relationship between telomerase deficiency and the generation of chromosomal translocations, a common characteristic of cancer cells. Telomerase deficiency increased the rate of formation of spontaneous translocations by homologous recombination involving telomere proximal sequences during crisis. However, telomerase deficiency also decreased the frequency of translocation formation following multiple HO-endonuclease catalyzed DNA double-strand breaks at telomere proximal or distal sequences before, during and after crisis. This decrease correlated with a sequestration of the central homologous recombination factor, Rad52, to telomeres determined by chromatin immuno-precipitation. This suggests that telomerase deficiency results in the sequestration of Rad52 to telomeres, limiting the capacity of the cell to repair double-strand breaks throughout the genome. Increased spontaneous translocation formation in telomerase-deficient yeast cells undergoing crisis is consistent with the increased incidence of cancer in elderly humans, as the majority of our cells lack telomerase. Decreased translocation formation by recombinational repair of double-strand breaks in telomerase-deficient yeast suggests that the reemergence of telomerase expression observed in many human tumors may further stimulate genome rearrangement. Thus, telomerase may exert a substantial effect on global genome stability, which may bear significantly on the appearance and progression of cancer in humans

Nucleotide Excision Repair, Genome Stability, and Human Disease: New Insight from Model Systems

Nucleotide excision repair (NER) is one of several DNA repair pathways that are universal throughout phylogeny. NER has a broad substrate specificity and is capable of removing several classes of lesions to the DNA, including those that accumulate upon exposure to UV radiation. The loss of this activity in NER-defective mutants gives rise to characteristic sensitivities to UV that, in humans, is manifested as a greatly elevated sensitivity to exposure to the sun. Xeroderma pigmentosum (XP), Cockaynes syndrome (CS), and trichothiodystrophy (TTD) are three, rare, recessively inherited human diseases that are linked to these defects. Interestingly, some of the symptoms in afflicted individuals appear to be due to defects in transcription, the result of the dual functionality of several components of the NER apparatus as parts of transcription factor IIH (TFIIH). Studies with several model systems have revealed that the genetic and biochemical features of NER are extraordinarily conserved in eukaryotes. One system that has been studied very closely is the budding yeast Saccharomyces cerevisiae. While many yeast NER mutants display the expected increases in UV sensitivity and defective transcription, other interesting phenotypes have also been observed. Elevated mutation and recombination rates, as well as increased frequencies of genome rearrangement by retrotransposon movement and recombination between short genomic sequences have been documented. The potential relevance of these novel phenotypes to disease in humans is discussed

RAD51C Germline Mutations in Breast and Ovarian Cancer Cases from High-Risk Families

BRCA1 and BRCA2 are the most well-known breast cancer susceptibility genes. Additional genes involved in DNA repair have been identified as predisposing to breast cancer. One such gene, RAD51C, is essential for homologous recombination repair. Several likely pathogenic RAD51C mutations have been identified in BRCA1- and BRCA2-negative breast and ovarian cancer families. We performed complete sequencing of RAD51C in germline DNA of 286 female breast and/or ovarian cancer cases with a family history of breast and ovarian cancers, who had previously tested negative for mutations in BRCA1 and BRCA2. We screened 133 breast cancer cases, 119 ovarian cancer cases, and 34 with both breast and ovarian cancers. Fifteen DNA sequence variants were identified; including four intronic, one 5′ UTR, one promoter, three synonymous, and six non-synonymous variants. None were truncating. The in-silico SIFT and Polyphen programs were used to predict possible pathogenicity of the six non-synonomous variants based on sequence conservation. G153D and T287A were predicted to be likely pathogenic. Two additional variants, A126T and R214C alter amino acids in important domains of the protein such that they could be pathogenic. Two-hybrid screening and immunoblot analyses were performed to assess the functionality of these four non-synonomous variants in yeast. The RAD51C-G153D protein displayed no detectable interaction with either XRCC3 or RAD51B, and RAD51C-R214C displayed significantly decreased interaction with both XRCC3 and RAD51B (p<0.001). Immunoblots of RAD51C-Gal4 activation domain fusion peptides showed protein levels of RAD51C-G153D and RAD51C-R214C that were 50% and 60% of the wild-type, respectively. Based on these data, the RAD51C-G153D variant is likely to be pathogenic, while the RAD51C- R214C variant is hypomorphic of uncertain pathogenicity. These results provide further support that RAD51C is a rare breast and ovarian cancer susceptibility gene

Variants of the human RAD52 gene confer defects in ionizing radiation resistance and homologous recombination repair in budding yeast

RAD52 is a structurally and functionally conserved component of the DNA double-strand break (DSB) repair apparatus from budding yeast to humans. We recently showed that expressing the human gene, HsRAD52 in rad52 mutant budding yeast cells can suppress both their ionizing radiation (IR) sensitivity and homologous recombination repair (HRR) defects. Intriguingly, we observed that HsRAD52 supports DSB repair by a mechanism of HRR that conserves genome structure and is independent of the canonical HR machinery. In this study we report that naturally occurring variants of HsRAD52, one of which suppresses the pathogenicity of BRCA2 mutations, were unable to suppress the IR sensitivity and HRR defects of rad52 mutant yeast cells, but fully suppressed a defect in DSB repair by single-strand annealing (SSA). This failure to suppress both IR sensitivity and the HRR defect correlated with an inability of HsRAD52 protein to associate with and drive an interaction between genomic sequences during DSB repair by HRR. These results suggest that HsRAD52 supports multiple, distinct DSB repair apparatuses in budding yeast cells and help further define its mechanism of action in HRR. They also imply that disruption of HsRAD52-dependent HRR in BRCA2-defective human cells may contribute to protection against tumorigenesis and provide a target for killing BRCA2-defective cancers

RAD59 and RAD1 cooperate in translocation formation by single-strand annealing in Saccharomyces cerevisiae

Studies in the budding yeast, Saccharomyces cerevisiae, have demonstrated that a substantial fraction of double-strand break repair following acute radiation exposure involves homologous recombination between repetitive genomic elements. We have previously described an assay in S. cerevisiae that allows us to model how repair of multiple breaks leads to the formation of chromosomal translocations by single-strand annealing (SSA) and found that Rad59, a paralog of the single-stranded DNA annealing protein Rad52, is critically important in this process. We have constructed several rad59 missense alleles to study its function more closely. Characterization of these mutants revealed proportional defects in both translocation formation and spontaneous direct-repeat recombination, which is also thought to occur by SSA. Combining the rad59 missense alleles with a null allele of RAD1, which encodes a subunit of a nuclease required for the removal of non-homologous tails from annealed intermediates, substantially suppressed the low frequency of translocations observed in rad1-null single mutants. These data suggest that at least one role of Rad59 in translocation formation by SSA is supporting the machinery required for cleavage of non-homologous tails

The charcoal trap: Miombo forests and the energy needs of people

<p>Abstract</p> <p>Background</p> <p>This study evaluates the carbon dioxide and other greenhouse gas fluxes to the atmosphere resulting from charcoal production in Zambia. It combines new biomass and flux data from a study, that was conducted in a <it>miombo </it>woodland within the Kataba Forest Reserve in the Western Province of Zambia, with data from other studies.</p> <p>Results</p> <p>The measurements at Kataba compared protected area (3 plots) with a highly disturbed plot outside the forest reserve and showed considerably reduced biomass after logging for charcoal production. The average aboveground biomass content of the reserve (Plots 2-4) was around 150 t ha^-1, while the disturbed plot only contained 24 t ha^-1. Soil carbon was not reduced significantly in the disturbed plot. Two years of eddy covariance measurements resulted in net ecosystem exchange values of -17 ± 31 g C m^-2y^-1, in the first and 90 ± 16 g C m^-2in the second year. Thus, on the basis of these two years of measurement, there is no evidence that the <it>miombo </it>woodland at Kataba represents a present-day carbon sink. At the country level, it is likely that deforestation for charcoal production currently leads to a per capita emission rate of 2 - 3 t CO₂y^-1. This is due to poor forest regeneration, although the resilience of <it>miombo </it>woodlands is high. Better post-harvest management could change this situation.</p> <p>Conclusions</p> <p>We argue that protection of <it>miombo </it>woodlands has to account for the energy demands of the population. The production at national scale that we estimated converts into 10,000 - 15,000 GWh y^-1of energy in the charcoal. The term "Charcoal Trap" we introduce, describes the fact that this energy supply has to be substituted when woodlands are protected. One possible solution, a shift in energy supply from charcoal to electricity, would reduce the pressure of forests but requires high investments into grid and power generation. Since Zambia currently cannot generate this money by itself, the country will remain locked in the charcoal trap such as many other of its African neighbours. The question arises whether and how money and technology transfer to increase regenerative electrical power generation should become part of a post-Kyoto process. Furthermore, better inventory data are urgently required to improve knowledge about the current state of the woodland usage and recovery. Net greenhouse gas emissions could be reduced substantially by improving the post-harvest management, charcoal production technology and/or providing alternative energy supply.</p

Rad51 Inhibits Translocation Formation by Non-Conservative Homologous Recombination in Saccharomyces cerevisiae

Chromosomal translocations are a primary biological response to ionizing radiation (IR) exposure, and are likely to result from the inappropriate repair of the DNA double-strand breaks (DSBs) that are created. An abundance of repetitive sequences in eukaryotic genomes provides ample opportunity for such breaks to be repaired by homologous recombination (HR) between non-allelic repeats. Interestingly, in the budding yeast, Saccharomyces cerevisiae the central strand exchange protein, Rad51 that is required for DSB repair by gene conversion between unlinked repeats that conserves genomic structure also suppresses translocation formation by several HR mechanisms. In particular, Rad51 suppresses translocation formation by single-strand annealing (SSA), perhaps the most efficient mechanism for translocation formation by HR in both yeast and mammalian cells. Further, the enhanced translocation formation that emerges in the absence of Rad51 displays a distinct pattern of genetic control, suggesting that this occurs by a separate mechanism. Since hypomorphic mutations in RAD51 in mammalian cells also reduce DSB repair by conservative gene conversion and stimulate non-conservative repair by SSA, this mechanism may also operate in humans and, perhaps contribute to the genome instability that propels the development of cancer

Msh2 Blocks an Alternative Mechanism for Non-Homologous Tail Removal during Single-Strand Annealing in Saccharomyces cerevisiae

Chromosomal translocations are frequently observed in cells exposed to agents that cause DNA double-strand breaks (DSBs), such as ionizing radiation and chemotherapeutic drugs, and are often associated with tumors in mammals. Recently, translocation formation in the budding yeast, Saccharomyces cerevisiae, has been found to occur at high frequencies following the creation of multiple DSBs adjacent to repetitive sequences on non-homologous chromosomes. The genetic control of translocation formation and the chromosome complements of the clones that contain translocations suggest that translocation formation occurs by single-strand annealing (SSA). Among the factors important for translocation formation by SSA is the central mismatch repair (MMR) and homologous recombination (HR) factor, Msh2. Here we describe the effects of several msh2 missense mutations on translocation formation that suggest that Msh2 has separable functions in stabilizing annealed single strands, and removing non-homologous sequences from their ends. Additionally, interactions between the msh2 alleles and a null allele of RAD1, which encodes a subunit of a nuclease critical for the removal of non-homologous tails suggest that Msh2 blocks an alternative mechanism for removing these sequences. These results suggest that Msh2 plays multiple roles in the formation of chromosomal translocations following acute levels of DNA damage

Multiple Pathways Promote Short-Sequence Recombination in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, null alleles of several DNA repair and recombination genes confer defects in recombination that grow more severe with decreasing sequence length, indicating that they are required for short-sequence recombination (SSR). RAD1 and RAD10, which encode the subunits of the structure-specific endonuclease Rad1/10, are critical for SSR. MRE11, RAD50, and XRS2, which encode the subunits of M/R/X, another complex with nuclease activity, are also crucially important. Genetic evidence suggests that Rad1/10 and M/R/X act on the same class of substrates during SSR. MSH2 and MSH3, which encode subunits of Msh2/3, a complex active during mismatch repair and recombination, are also important for SSR but play a more restricted role. Additional evidence suggests that SSR is distinct from nonhomologous end joining and is superimposed upon basal homologous recombination