Biomedical subjects
Publications and source records attributed to L Guarente.
As normal humans age, telomeres shorten in tissues that contain dividing cells, and this has been proposed both as a cause of ageing and as a tumor-suppressor mechanism. The surprising finding that cells from individuals with the rare inherited disorder dyskeratosis congenita (DKC) have reduced levels of telomerase and shortened telomeres might provide the first direct genetic test of the function of telomeres in intact humans.
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Werner's syndrome (WS) is a human disease with manifestations resembling premature aging. The gene defective in WS, WRN, encodes a DNA helicase. Here, we describe the generation of mice bearing a mutation that eliminates expression of the C terminus of the helicase domain of the WRN protein. Mutant mice are born at the expected Mendelian frequency and do not show any overt histological signs of accelerated senescence. These mice are capable of living beyond 2 years of age. Cells from these animals do not show elevated susceptibility to the genotoxins camptothecin or 4-NQO. However, mutant fibroblasts senesce approximately one passage earlier than controls. Importantly, WRN(-/-);p53(-/-) mice show an increased mortality rate relative to WRN(+/-);p53(-/-) animals. We consider possible models for the synergy between p53 and WRN mutations for the determination of life span.
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The SIR genes are determinants of life span in yeast mother cells. Here we show that life span regulation by the Sir proteins is independent of their role in nonhomologous end joining. The short life span of a sir3 or sir4 mutant is due to the simultaneous expression of a and alpha mating-type information, which indirectly causes an increase in rDNA recombination and likely increases the production of extrachromosomal rDNA circles. The short life span of a sir2 mutant also reveals a direct failure to repress recombination generated by the Fob1p-mediated replication block in the rDNA. Sir2p is a limiting component in promoting yeast longevity, and increasing the gene dosage extends the life span in wild-type cells. A possible role of the conserved SIR2 in mammalian aging is discussed.
Saccharomyces cerevisiae mother cells undergo an aging program that includes morphologic changes, sterility, redistribution of the Sir transcriptional silencing complex from HM loci and telomeres to the nucleolus, alterations in nucleolar architecture, and accumulation of extrachromosomal ribosomal DNA circles (ERCs). We report here that cells starved for nutrients during prolonged periods in stationary phase show a decrease in generational lifespan when they reenter the cell cycle. This shortened lifespan is not transmitted to progeny cells, indicating that it is not due to irreversible genetic damage. The decrease in the lifespan is accompanied by all of the changes of accelerated aging with the notable exception that ERC accumulation is not augmented compared with generation-matched, nonstarved cells. These results suggest a number of models, including one in which starvation reveals a component of aging that works in parallel with the accumulation of ERCs. Stationary-phase yeast cells may be a useful system for identifying factors that affect aging in other nondividing eukaryotic cells.
The yeast Sir2/3/4p complex is found in abundance at telomeres, where it participates in the formation of silent heterochromatin and telomere maintenance. Here, we show that Sir3p is released from telomeres in response to DNA double-strand breaks (DSBs), binds to DSBs, and mediates their repair, independent of cell mating type. Sir3p relocalization is S phase specific and, importantly, requires the DNA damage checkpoint genes MEC1 and RAD9. MEC1 is a homolog of ATM, mutations in which cause ataxia telangiectasia (A-T), a disease characterized by various neurologic and immunologic abnormalities, a predisposition for cancer, and a cellular defect in repair of DSBs. This novel mode by which preformed DNA repair machinery is mobilized by DNA damage sensors may have implications for human diseases resulting from defective DSB repair.
A cause of aging in yeast is the accumulation of circular species of ribosomal DNA (rDNA) arising from the 100-200 tandemly repeated copies in the genome. We show here that mutation of the FOB1 gene slows the generation of these circles and thus extends life span. Fob1p is known to create a unidirectional block to replication forks in the rDNA. We show that Fob1p is a nucleolar protein, suggesting a direct involvement in the replication fork block. We propose that this block can trigger aging by causing chromosomal breaks, the repair of which results in the generation of rDNA circles. These findings may provide a novel link between metabolic rate and aging in yeast and, perhaps, higher organisms.
The yeast Sir protein complex has been implicated in transcriptional silencing and suppression of recombination. The Sir complex creates structured chromosomal domains at telomeres, silent mating-type loci and ribosomal DNA to invoke these functional states. Mechanistic insights into the function of Sir proteins implicate a range of activities in yeast, including repair of DNA double-strand breaks, regulation of the mitotic cell cycle, meiosis and ageing. I speculate that the Sir proteins may be capable of enzymatic modification of chromatin and other substrates, which enables them to carry out a broad range of cellular functions.
HAP1 is a yeast transcriptional activator that binds with equal affinity to the dissimilar upstream activation sequences UAS1 and UAS(CYC7), but activates transcription differentially when bound to each site. HAP1-18 harbors an amino acid change in the DNA binding domain. While binding UAS1 poorly, HAP1-18 binds UAS(CYC7) with wild-type properties and activates transcription at elevated levels relative to HAP1. We have determined the structure of HAP1-18-UAS(CYC7) and have compared it to HAP1-UAS(CYC7). Unexpectedly, the single amino acid substitution in HAP1-18 nucleates a significantly altered hydrogen bond interface between the protein and DNA resulting in DNA conformational changes and an ordering of one N-terminal arm of the protein dimer along the DNA minor groove. These observations, together with a large subset of transcriptionally defective mutations in the HAP1 DNA-binding domain that map to the HAP1-DNA interface, suggest that protein-DNA interactions may have direct allosteric effects on transcriptional activation.
HAP1 is a member of a family of fungal transcription factors that contain a Zn2Cys6 binuclear cluster domain and bind as homodimers to sequences containing two DNA half sites. We have determined the 2.5 A crystal structure of HAP1 bound to a cognate upstream activation sequence from the CYC7 gene. The structure reveals that HAP1 is bound in a dramatically asymmetric manner to the DNA target. This asymmetry aligns the Zn2Cys6 domains in a tandem head-to-tail fashion to contact two DNA half sites, positions an N-terminal arm of one of the protein subunits to interact with the inter-half site base pairs in the DNA minor groove, and suggests a mechanism by which DNA-binding facilitates asymmetric dimerization by HAP1. Comparisons with the DNA complexes of the related GAL4, PPR1 and PUT3 proteins illustrate how a conserved protein domain can be reoriented to recognize DNA half sites of different polarities and how homodimeric proteins adopt dramatically asymmetric structures to recognize cognate DNA targets.
A cause of aging in Saccharomyces cerevisiae is the accumulation of extrachromosomal ribosomal DNA circles (ERCs). Introduction of an ERC into young mother cells shortens life span and accelerates the onset of age-associated sterility. It is important to understand the process by which ERCs are generated. Here, we demonstrate that homologous recombination is necessary for ERC formation. rad52 mutant cells, defective in DNA repair through homologous recombination, do not accumulate ERCs with age, and mutations in other genes of the RAD52 class have varying effects on ERC formation. rad52 mutation leads to a progressive delocalization of Sir3p from telomeres to other nuclear sites with age and, surprisingly, shortens life span. We speculate that spontaneous DNA damage, perhaps double-strand breaks, causes lethality in mutants of the RAD52 class and may be an initial step of aging in wild-type cells.
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Werner Syndrome (WS) is a human genetic disorder with many features of premature aging. The gene defective in WS (WRN) has been cloned and encodes a protein homologous to several helicases, including Escherichia coli RecQ, the human Bloom syndrome protein (BLM), and Saccharomyces cerevisiae Sgs1p. To better define the function of WRN protein we have determined its subcellular localization. Indirect immunofluorescence using polyclonal anti-human WRN shows a predominant nucleolar localization. Studies of WRN mutant cells lines confirmed the specificity of antibody recognition. No difference was seen in the subcellular localization of the WRN protein in a variety of normal and transformed human cell lines, including both carcinomas and sarcomas. The nucleolar localization of human WRN protein was supported by the finding that upon biochemical subcellular fractionation, WRN protein is present in an increased concentration in a subnuclear fraction enriched for nucleolar proteins. We have also determined the subcellular localization of the mouse WRN homologue (mWRN). In contrast to human WRN protein, mWRN protein is present diffusely throughout the nucleus. Understanding the function of WRN in these organisms of vastly differing lifespan may yield new insights into the mechanisms of lifespan determination.
Reactivation of telomerase in cultured human cells extends their replicative life span beyond the Hayflick limit. How telomere shortening triggers cell senescence and whether it contributes to aging in vivo are under investigation. Studies in yeast have revealed another site critical to cellular aging: the nucleolus. The accumulation of ribosomal DNA circles is a cause of aging in this organism. The possible relevance of this mechanism to human aging is also being considered.
The life cycle of many organisms involves a progressive decline in fitness and fecundity with age, and yeast is no exception. Many theories have been proposed to explain the mortality of yeast cells, including the increase in cell size and accumulation of bud scars on the cell surface. None of these has survived closed scrutiny. However, recent discoveries might have validated one aging model in which the triggering of a molecular aging clock results in the replication and accumulation of a senescence factor that eventually overwhelms old cells.
The past year has confirmed the great potential of the yeast Saccharomyces cerevisiae as a model to study aging. Ground breaking papers have revealed similarities between aging in yeast and in mammals, and have identified genetic instability of the ribosomal DNA array as the first known cause of aging in yeast cells.