Defective DNA Repair and Increased Genomic Instability in Artemis-deficient Murine Cells

In developing lymphocytes, the recombination activating gene endonuclease cleaves DNA between V, D, or J coding and recombination signal (RS) sequences to form hairpin coding and blunt RS ends, which are fused to form coding and RS joins. Nonhomologous end joining (NHEJ) factors repair DNA double strand breaks including those induced during VDJ recombination. Human radiosensitive severe combined immunodeficiency results from lack of Artemis function, an NHEJ factor with in vitro endonuclease/exonuclease activities. We inactivated Artemis in murine embryonic stem (ES) cells by targeted mutation. Artemis deficiency results in impaired VDJ coding, but not RS, end joining. In addition, Artemis-deficient ES cells are sensitive to a radiomimetic drug, but less sensitive to ionizing radiation. VDJ coding joins from Artemis-deficient ES cells, which surprisingly are distinct from the highly deleted joins consistently obtained from DNA-dependent protein kinase catalytic subunit–deficient ES cells, frequently lack deletions and often display large junctional palindromes, consistent with a hairpin coding end opening defect. Strikingly, Artemis-deficient ES cells have increased chromosomal instability including telomeric fusions. Thus, Artemis appears to be required for a subset of NHEJ reactions that require end processing. Moreover, Artemis functions as a genomic caretaker, most notably in prevention of translocations and telomeric fusions. As Artemis deficiency is compatible with human life, Artemis may also suppress genomic instability in humans

Calorie restriction alters mitochondrial protein acetylation

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72130/1/j.1474-9726.2009.00503.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/72130/2/ACEL_503_sm_FigS1.pd

C. elegans SIRT6/7 Homolog SIR-2.4 Promotes DAF-16 Relocalization and Function during Stress

FoxO transcription factors and sirtuin family deacetylases regulate diverse biological processes, including stress responses and longevity. Here we show that the Caenorhabditis elegans sirtuin SIR-2.4—homolog of mammalian SIRT6 and SIRT7 proteins—promotes DAF-16–dependent transcription and stress-induced DAF-16 nuclear localization. SIR-2.4 is required for resistance to multiple stressors: heat shock, oxidative insult, and proteotoxicity. By contrast, SIR-2.4 is largely dispensable for DAF-16 nuclear localization and function in response to reduced insulin/IGF-1-like signaling. Although acetylation is known to regulate localization and activity of mammalian FoxO proteins, this modification has not been previously described on DAF-16. We find that DAF-16 is hyperacetylated in sir-2.4 mutants. Conversely, DAF-16 is acetylated by the acetyltransferase CBP-1, and DAF-16 is hypoacetylated and constitutively nuclear in response to cbp-1 inhibition. Surprisingly, a SIR-2.4 catalytic mutant efficiently rescues the DAF-16 localization defect in sir-2.4 null animals. Acetylation of DAF-16 by CBP-1 in vitro is inhibited by either wild-type or mutant SIR-2.4, suggesting that SIR-2.4 regulates DAF-16 acetylation indirectly, by preventing CBP-1-mediated acetylation under stress conditions. Taken together, our results identify SIR-2.4 as a critical regulator of DAF-16 specifically in the context of stress responses. Furthermore, they reveal a novel role for acetylation, modulated by the antagonistic activities of CBP-1 and SIR-2.4, in modulating DAF-16 localization and function

Functional Interaction of H2AX, NBS1, and p53 in ATM-Dependent DNA Damage Responses and Tumor Suppression

Ataxia-telangiectasia (A-T) mutated (ATM) kinase signals all three cell cycle checkpoints after DNA double-stranded break (DSB) damage. H2AX, NBS1, and p53 are substrates of ATM kinase and are involved in ATM-dependent DNA damage responses. We show here that H2AX is dispensable for the activation of ATM and p53 responses after DNA DSB damage. Therefore, H2AX functions primarily as a downstream mediator of ATM functions in the parallel pathway of p53. NBS1 appears to function both as an activator of ATM and as an adapter to mediate ATM activities after DNA DSB damage. Phosphorylation of ATM and H2AX induced by DNA DSB damage is normal in NBS1 mutant/mutant (NBS1(m/m)) mice that express an N-terminally truncated NBS1 at lower levels. Therefore, the pleiotropic A-T-related systemic and cellular defects observed in NBS1(m/m) mice are due to the disruption of the adapter function of NBS1 in mediating ATM activities. While H2AX is required for the irradiation-induced focus formation of NBS1, our findings indicate that NBS1 and H2AX have distinct roles in DNA damage responses. ATM-dependent phosphorylation of p53 and p53 responses are largely normal in NBS1(m/m) mice after DNA DSB damage, and p53 deficiency greatly facilitates tumorigenesis in NBS1(m/m) mice. Therefore, NBS1, H2AX, and p53 play synergistic roles in ATM-dependent DNA damage responses and tumor suppression

Rad54 and DNA Ligase IV cooperate to maintain mammalian chromatid stability

Nonhomologous end joining (NHEJ) and homologous recombination (HR) represent the two major pathways of DNA double-strand break (DSB) repair in eukaryotic cells. NHEJ repairs DSBs by ligation of cognate broken ends irrespective of homologous flanking sequences, whereas HR repairs DSBs using an undamaged homologous template. Although both NHEJ and HR have been clearly implicated in the maintenance of genome stability, how these apparently independent and mechanistically distinct pathways are coordinated remains largely unexplored. To investigate the relationship between HR and NHEJ modes of DSB repair, we generated cells doubly deficient for the NHEJ factor DNA Ligase IV (Lig4) and the HR factor Rad54. We show that Lig4 and Rad54 cooperate to support cellular proliferation, repair spontaneous DSBs, and prevent chromosome and single chromatid aberrations. These findings demonstrate a role for NHEJ in the repair of DSBs that occur spontaneously during or after DNA replication, and reveal overlapping functions for NHEJ and Rad54-dependent HR in the repair of such DSBs

Calorie Restriction Alters Mitochondrial Protein Acetylation

Calorie restriction (CR) increases lifespan in organisms ranging from budding yeast through mammals. Mitochondrial adaptation represents a key component of the response to CR. Molecular mechanisms underlying this adaptation are largely unknown. Here we show that lysine acetylation of mitochondrial proteins is altered during CR in a tissue-specific fashion. Via large-scale mass spectrometry screening, we identify 72 candidate proteins involved in a variety of metabolic pathways with altered acetylation during CR. Mitochondrial acetylation changes may play an important role in the pro-longevity CR response

SIR-2.4 is required for optimal DAF-16–dependent gene expression.

<p>Wild-type N2 animals fed on either vector control or <i>sir-2.4</i> RNAi bacteria from the time of hatching were exposed to 10 mM H₂O₂ for 80 min. Relative mRNA levels of SOD-3, HSP-16.1, DOD-3, DOD-24, C32H11.4, and INS-7 were measured by quantitative RT-PCR and the means of three different sample sets are shown. Relative mRNA levels were normalized against ACT-1 (beta-actin). Error bars: ± STD. Statistical significance as determined by two-tailed t-test is shown in the table below; significant differences are represented in black font.</p