Our overall goal is to gain a better understanding of mechanisms underlying the DNA damage response (DDR) and maintenance of genomic integrity. We will study the function of the CtIP tumor suppressor protein, conserved in all eukaryotes. Specifically, we will investigate the role of CtIP in DNA double-strand break (DSB) end processing, and ATR activation, and its function in the process of 5'->3'resection at a DSB. Our preliminary studies in the Xenopus egg extract indicate that CtIP acts downstream of ATM in response to DSBs, and promotes DSB 5'end resection to generate ssDNA, triggering ATR and checkpoint activation. We will use the Xenopus extract and human cell lines, coupled with biochemical studies, to gain mechanistic understanding of the checkpoint role of CtIP and the process of DSB 5'end resection. In particular, we will ask if CtIP is important for processing DSBs with blocked ends, and if CtIP has intrinsic nuclease activity required for 5'->3'resection. We will determine the functional relationships between CtIP, ATM and the Mre11-Rad50- Nbs1 complex in resection. We have discovered a conserved class of RING finger E3 ubiquitin ligases that specifically target SUMOylated proteins. RNF4 family proteins (S. pombe Rfp1/Rfp2-Slx8, or human RNF4) recognize SUMOylated proteins via an N-terminal SUMO-interacting motif (SIM), and ubiquitinate SUMOylated proteins in vitro, dependent on the SIM and RING domains. RNF4 orthologues are implicated in maintenance of genomic integrity in several organisms. To elucidate the physiological roles of RNF4 family proteins, we will identify in vivo targets for RNF4 family proteins using GST-SIM and tagged-SUMO to affinity purify SUMOylated proteins from yeast and human cells, followed by validation and functional characterization of identified proteins as RNF4 targets in vivo and in vitro. We will test candidate proteins implicated by genetic interactions with Rfp1/2 in S. pombe and through physical interaction with RNF4 as targets, including TRIM family proteins. We will determine the significance of the unique structural elements in the multi-SIM and RING domains of RNF4 family proteins in target recognition, and test the model that they recognize poly- SUMOylated proteins. Overall, we expect to learn how RNF4 family proteins function in the DNA damage response and in maintaining genomic integrity. The DNA damage response pathway involves activation of a series of protein kinases, including ATM, ATR, and Chk1/Chk2. To learn more about the network of events downstream of DNA damage, we will use SILAC labeling, phosphoprotein/peptide enrichment, and quantitative mass spectrometry to identify and functionally characterize novel protein phosphorylation sites in mammalian cells induced by DNA damage. For this purpose, we will use a synchronized HeLa cell system where DNA damage is induced with MMS;samples taken at various times and doses of MMS will be analyzed by mass spectrometry for novel phosphorylation events, whose role in the DDR will subsequently be determined.

Public Health Relevance

The studies proposed in this application seek to elucidate at the molecular level how cells respond to DNA damage and maintain a stable genome, which are key to understanding how cells safeguard against the development of cancer-causing mutations. Since many cancer therapeutics kill cancer cells by eliciting DNA damage, our studies will also provide insights into the sensitivity of cancer cells to these agents, and suggest ways to improve their use.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA080100-14
Application #
8268540
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Hildesheim, Jeffrey
Project Start
1999-06-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
14
Fiscal Year
2012
Total Cost
$706,686
Indirect Cost
$333,568
Name
Salk Institute for Biological Studies
Department
Type
DUNS #
078731668
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Zheng, Xinde; Boyer, Leah; Jin, Mingji et al. (2016) Alleviation of neuronal energy deficiency by mTOR inhibition as a treatment for mitochondria-related neurodegeneration. Elife 5:
Zheng, Xinde; Boyer, Leah; Jin, Mingji et al. (2016) Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation. Elife 5:
Heimbucher, Thomas; Hunter, Tony (2015) The C. elegans Ortholog of USP7 controls DAF-16 stability in Insulin/IGF-1-like signaling. Worm 4:e1103429
Heimbucher, Thomas; Liu, Zheng; Bossard, Carine et al. (2015) The Deubiquitylase MATH-33 Controls DAF-16 Stability and Function in Metabolism and Longevity. Cell Metab 22:151-63
Fuhs, Stephen Rush; Meisenhelder, Jill; Aslanian, Aaron et al. (2015) Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation. Cell 162:198-210
Wilkinson, Deepti S; Jariwala, Jinel S; Anderson, Ericka et al. (2015) Phosphorylation of LC3 by the Hippo kinases STK3/STK4 is essential for autophagy. Mol Cell 57:55-68
Carrano, Andrea C; Dillin, Andrew; Hunter, Tony (2014) A Krüppel-like factor downstream of the E3 ligase WWP-1 mediates dietary-restriction-induced longevity in Caenorhabditis elegans. Nat Commun 5:3772
Zhang, Youwei; Hunter, Tony (2014) Roles of Chk1 in cell biology and cancer therapy. Int J Cancer 134:1013-23
Zheng, Xinde; Hunter, Tony (2014) Pink1, the first ubiquitin kinase. EMBO J 33:1621-3
Aslanian, Aaron; Yates 3rd, John R; Hunter, Tony (2014) Mass spectrometry-based quantification of the cellular response to methyl methanesulfonate treatment in human cells. DNA Repair (Amst) 15:29-38

Showing the most recent 10 out of 37 publications