SUMMARY Defective DNA repair by homologous recombination (HR) is thought to be a major contributor to tumorigenesis in individuals carrying Brca1 mutations. Here we show that DNA breaks in Brca1-deficient cells are aberrantly joined into complex chromosome rearrangements by a process dependent on the non-homologous end joining (NHEJ) factors, 53BP1 and DNA Ligase 4. Loss of 53BP1 alleviates hypersensitivity of Brca1 mutant cells to PARP inhibition and restores error-free repair by HR. Mechanistically, 53BP1 deletion promotes ATM-dependent processing of broken DNA ends to produce recombinogenic single-stranded DNA competent for HR. In contrast, Lig4 deficiency does not rescue the HR defect in Brca1 mutant cells, but prevents the joining of chromatid breaks into chromosome rearrangements. Our results illustrate that HR and NHEJ compete to process DNA breaks that arise during DNA replication, and that shifting the balance between these pathways can be exploited to selectively protect or kill cells harboring Brca1 mutations.
We demonstrate a role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy. In particular, transient increased expression of Sirt1 is sufficient to stimulate basal rates of autophagy. In addition, we show that Sirt1 ؊/؊ mouse embryonic fibroblasts do not fully activate autophagy under starved conditions. Reconstitution with wild-type but not a deacetylase-inactive mutant of Sirt1 restores autophagy in these cells. We further demonstrate that Sirt1 can form a molecular complex with several essential components of the autophagy machinery, including autophagy genes (Atg)5, Atg7, and Atg8. In vitro, Sirt1 can, in an NAD-dependent fashion, directly deacetylate these components. The absence of Sirt1 leads to markedly elevated acetylation of proteins known to be required for autophagy in both cultured cells and in embryonic and neonatal tissues. Finally, we show that Sirt1 ؊/؊ mice partially resemble Atg5 ؊/؊ mice, including the accumulation of damaged organelles, disruption of energy homeostasis, and early perinatal mortality. Furthermore, the in utero delivery of the metabolic substrate pyruvate extends the survival of Sirt1 ؊/؊ pups. These results suggest that the Sirt1 deacetylase is an important in vivo regulator of autophagy and provide a link between sirtuin function and the overall cellular response to limited nutrients. mitochondria ͉ starvation ͉ acetylation ͉ aging M acroautophagy, referred to hereafter as autophagy, is an intracellular process that allows for the degradation of proteins and organelles (1-3). Morphologically, autophagy is characterized by the formation of a double-membrane structure termed the autophagosome. In yeast, the process of autophagy can be stimulated by the withdrawal of various nutrients, and evidence suggests that at least 16 separate autophagy genes (Atg) are ultimately required for the formation of the autophagosome and the subsequent induction of autophagy (1-3). Mammalian cells can also respond to nutrient withdrawal by inducing autophagy. Genetic studies have suggested that autophagy is required to maintain the animal's energetic needs during the small window of time immediately after birth but before neonates can get milk nutrients from their mothers (4). In addition, two recent studies have demonstrated that brain conditional knockouts of either Atg7 or Atg5 significantly accelerated the development of neuropathologies usually restricted to older animals (5, 6).The sirtuins are a family of NAD-dependent deacetylases that have been linked to the regulation of life span. Increased expression of Sir2 can extend the life span of model organisms, and under certain experimental conditions, it appears that the activity of Sir2 is required for the life-extending benefits of caloric restriction in organisms such as yeast and flies (7). As opposed to these relatively simple organisms, mammalian species have seven different sirtuin family members (8, 9). The closest relative of yeast Sir2 is termed Sirt1 in mammalian cells. We have demonstrated that mice subjected t...
Summary In lower eukaryotes, Sir2 serves as a histone deacetylase and is implicated in chromatin silencing, longevity and genome stability. Here we mutated the SIRT1 gene, a homolog of yeast Sir2, in mice to study its function. We showed that a majority of SIRT1-null embryos died between E9.5–E14.5, displaying altered histone modification, impaired DNA damage response, and reduced ability to repair DNA damage. We demonstrated that SIRT1+/−;p53+/− mice developed tumors in multiple tissues, whereas activation of SIRT1 by resveratrol treatment reduced tumorigenesis. Finally, we showed that many human cancers exhibited reduced level of SIRT1 than their normal controls. Thus, SIRT1 acts as a tumor suppressor through its role in DNA damage response, genome integrity, and tumor suppression. Significance SIRT1 has diverse roles in various biological processes, including caloric restriction that causes changes in glucose metabolism and lifespan. The role of SIRT1 in cancer is currently under debate due to some recent different findings. It is known that calorie restriction, which activates SIRT1, extends lifespan and inhibits tumorigenesis. On the other hand, SIRT1 deacetylates p53 to decrease its activity. It was therefore hypothesized increased SIRT1 activity, although it extends lifespan, may elevate cancer risk. Here we demonstrate SIRT1 plays an important role in DNA damage response and genome integrity by maintaining proper chromatin structure and DNA damage repair foci formation. We further show that SIRT1 serves as a tumor suppressor in mice and in some types of human cancers.
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