Tanya Paull studies how double-strand breaks in DNA are recognized and repaired in eukaryotic cells and how oxidative stress induces signaling pathways that regulate cell growth and cell death.
Roles of the MRN Complex in the DNA Damage Response
The repair of double-strand breaks (DSBs) in chromosomal DNA occurs through recombination, either through nonhomologous mechanisms (NHEJ) or through homologous mechanisms that require replication from an intact template. Correct repair of DSBs in cells is essential for the maintenance of genomic stability, and errors in these pathways predispose mammalian organisms to cancer.
The Mre11/Rad50/Nbs1(Xrs2) [MRN(X)] complex is one of the first enzyme complexes to recognize and localize to a DSB in eukaryotic cells. MRN(X)-deficient cells exhibit reduced levels of DSB processing in vivo and are completely unable to process DSBs during meiosis when chromosomal breaks are made by the Spo11 protein. Genetic experiments have also identified other cellular factors that act in cooperation with MRN(X) to process DSBs in eukaryotic cells, including the Sae2 (CtIP/Ctp1) endonuclease, the Exo1 exonuclease, and the Sgs1 helicase.
Our studies investigate the biochemical basis of the involvement of MRN(X) and Sae2/CtIP in the processing of 5’ strands at DSBs using purified proteins in vitro, and characterize how posttranslational modifications of these factors affect their activities. We also investigate the functional effects of mutations in DSB repair factors in vivo in budding yeast and in human cells, examining how catalytic mutations and inhibition of posttranslational modifications affect cell cycle progression, DNA repair, and DNA damage signaling. The summer project will likely involve analysis of MRN(X) and Sae2/CtIP catalytic functions.
The ATM Protein Kinase and Regulation of Redox Homeostasis
The ataxia-telangiectasia mutated (ATM) kinase initiates a rapid cascade of protein phosphorylation in response to double-strand breaks in chromosomal DNA. We have also observed robust ATM activation in human cell lines and with purified protein in vitro in response to oxidative stress, in the absence of DNA damage. Thus, at least two independent mechanisms exist for the activation of ATM kinase activity in cells: one that is dependent on the Mre11/Rad50/Nbs1 (MRN) complex and the presence of linear DNA ends, and a separate pathway that is dependent only on the oxidation state of ATM. The oxidized form of ATM is a disulfide-crosslinked dimer containing several disulfide bonds, and mutation of a critical cysteine residue or the C-terminus of the FATC domain of ATM specifically blocks activation via the oxidation pathway, both in vitro with purified components and in human cells.
In this project, we are further investigating the role of ATM signaling in the maintenance of redox homeostasis in mammalian cells, through global proteomics screens, metabolite analysis, and analysis of signaling pathways in cells deficient in ATM. Evidence from diverse sources suggests that ATM is a critical component of redox control in human cells, such that loss of the kinase results in changes in insulin signaling, mitochondrial biogenesis, and metabolism. Identification of key targets of ATM in response to oxidative stress is essential for understanding the role of this kinase in these varied contexts.