The long-term goal of the proposed project is to understand the molecular mechanisms underlying age- related genomic instability. Genomic rearrangements accumulate in aging tissues leading to dysregulation of global transcription patterns and increase in cancer incidence. Aberrant repair of DNA double-stranded breaks (DSB) by nonhomologous end joining pathway (NHEJ) is responsible for the majority of the genomic rearrangements. NHEJ is the major DSB repair pathway in mammals, which typically leads to small deletions, but may also cause large genome rearrangements. Our preliminary data suggests that NHEJ efficiency and accuracy declines with age and during replicative senescence. Furthermore, we found that SIRT6, a mammalian homolog of yeast longevity gene Sir2, promotes DSB repair under oxidative stress and is able to complement senescence-related decline in repair. Despite the apparent role of NHEJ in aging and cancer age-related changes in NHEJ have not been systematically analyzed in vivo, and the mechanisms leading to age-related dysfunction of NHEJ are poorly understood. In the previous period of support we created a knock-in mouse model for analysis of age-related changes in NHEJ. This is the first mouse model that allows induction of DNA breaks and monitoring the efficiency and fidelity of NHEJ in vivo. This application seeks to utilize this mouse model to analyze age-related changes in NHEJ in different tissues, examine the role of SIRT6 in controlling the function of NHEJ, and test whether life-extending interventions act by stimulating NHEJ. We will pursue the following Specific Aims:
Aim 1 : Examine age-related changes in NHEJ in vivo. Here we will test the hypothesis that NHEJ becomes less efficient and more error-prone during in vivo aging. We will use our newly developed NHEJ reporter mouse to examine the efficiency and fidelity of NHEJ in a panel of tissues in young and aged mice.
Aim 2 : Test the hypothesis that SIRT6 serves as a regulator of DSB repair in response to stress and aging. Our preliminary data show that in cultured cells SIRT6 overexpression greatly stimulates DSB repair under conditions of oxidative stress. Our data also suggest SIRT6 acts on DSB repair by mono-ADP- ribosylating PARP1. Here we aim to expand these observations in an in vivo system using the NHEJ reporter mice and PARP1-/- mice.
Aim 3 : Test whether NHEJ is stimulated by rapamycin treatment or dietary restriction (DR). We hypothesize that decline of NHEJ function contributes to age-related genomic instability and functional decline. Both DR and rapamycin extend mammalian lifespan and have anticancer effect. Furthermore, DR increases SIRT6 levels. We, therefore hypothesize that these interventions stimulate DNA repair by NHEJ. To test this hypothesis we will subject NHEJ reporter mice to DR or rapamycin treatment for 1-8 months, and examine their NHEJ.
Genomes become unstable with age leading to functional decline of organs and tissues and increased incidence of cancer. Our goal is to understand the mechanisms of this instability using a unique mouse model that allows measuring the efficiency of DNA repair in vivo. In the long term, these studies will help develop novel ways to stabilize the aging genome, and prevent cancer.
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|Tan, Li; Ke, Zhonghe; Tombline, Gregory et al. (2017) Naked Mole Rat Cells Have a Stable Epigenome that Resists iPSC Reprogramming. Stem Cell Reports 9:1721-1734|
|Tian, Xiao; Seluanov, Andrei; Gorbunova, Vera (2017) Molecular Mechanisms Determining Lifespan in Short- and Long-Lived Species. Trends Endocrinol Metab 28:722-734|
|Hewitt, Graeme; Carroll, Bernadette; Sarallah, Rezazadeh et al. (2016) SQSTM1/p62 mediates crosstalk between autophagy and the UPS in DNA repair. Autophagy 12:1917-1930|
|Koschmann, Carl; Calinescu, Anda-Alexandra; Nunez, Felipe J et al. (2016) ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma. Sci Transl Med 8:328ra28|
|Gorbunova, Vera; Rezazadeh, Sarallah; Seluanov, Andrei (2016) Dangerous Entrapment for NRF2. Cell 165:1312-1313|
|Patrick, Alison; Seluanov, Michael; Hwang, Chaewon et al. (2016) Sensitivity of primary fibroblasts in culture to atmospheric oxygen does not correlate with species lifespan. Aging (Albany NY) 8:841-7|
|Ma, Siming; Upneja, Akhil; Galecki, Andrzej et al. (2016) Cell culture-based profiling across mammals reveals DNA repair and metabolism as determinants of species longevity. Elife 5:|
|Van Meter, Michael; Simon, Matthew; Tombline, Gregory et al. (2016) JNK Phosphorylates SIRT6 to Stimulate DNA Double-Strand Break Repair in Response to Oxidative Stress by Recruiting PARP1 to DNA Breaks. Cell Rep 16:2641-2650|
|Gorbunova, Vera; Seluanov, Andrei (2016) DNA double strand break repair, aging and the chromatin connection. Mutat Res 788:2-6|
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