The DNA damage Response (DDR) is a regulatory network that coordinates cellular processed in response to DNA damage and DNA replication stress. In addition, it directly orchestrates DNA repair choices because senses different DNA damage structures and transduces that information in cis to activate specific an appropriate repair options, thereby optimizing repair. The importance of the DDR is highlighted by the numerous cancer redisposition syndromes that result from its inactivation, including hereditary breast cancer resulting from BRCA1/2 mutation, AT, XP, and numerous others. Furthermore, targeted inhibition of the DDR can be exploited to enhance tumor sensitivity to radiation and genotoxic chemotherapy as well a synthetic lethality with the successful treatment of BRCA1/2-deficient tumors with PARP inhibitors. In the last 20 years we and others have investigated the composition of the central sensing and signaling apparatus used to detect and respond to genotoxic stress, first in yeast and more recently in mammals. This has revealed a conserved core of sensing and signaling proteins between yeast and mammals, just as there has been for classical DNA repair pathways. However, our analysis of substrates of this DDR kinase cascade has revealed an extremely diverse set of proteins and functions contacted by the DDR in mammals and the great majority of these activities are not conserved in yeast. Our analysis of ATM and ATR substrates and our and other analyses of non-ATM/ATR regulated phosphorylation events have implicated over 1000 proteins in the DDR the vast majority of which have no previous links to the DDR and have no yeast counterparts. Therefor there are likely to be many new components of the DDR to be discovered in mammals and it is critical that we set out to identify these factors in order to generate a complete understanding of the DDR and its significance in cellular and organismal physiology. Toward this end, we have developed sophisticated genetic tools that allow us to employ RNAi to find new protein candidates involved in promoting survival in response to DN damage. We have performed three preliminary screens to find new DDR candidate proteins that will serve at the basis for AIM1 and AIM2. In these AIMs we propose to carry out exhaustive validation of the candidate to identify bona fide new DDR proteins and will follow up on a candidate protein already validated. I addition we have also developed cutting edge proteomics methods to quantitatively detect post-translation modifications in response to DNA damage that form the basis of AIM3. In this AIMs we propose to identify new DDR proteins that are modified by ubiquitin and will follow one such protein that is known to regulate DNA damage foci formation. We also propose to use new proteomic and in situ localization screening to identify new proteins that localize to sites of DNA damage in AIM4. These innovative methods will allow us to deeply probe the layers of the DDR to identify and prioritize new proteins and to then probe the function of these proteins in the DDR using approaches we have pioneered for so many years.

Public Health Relevance

The DNA damage response pathway is critical to the maintenance of genomic integrity which is critical not only to prevent cancer but also many human diseases such as immunological and neurological developmental diseases, as well as playing a key role in human aging. Here we propose to systematically identify new genes involved in the human DNA damage response. These genes will allow us to have a deeper understanding of this global response and will guide us in understanding human disease and aging.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01GM044664-26
Application #
8720008
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Willis, Kristine Amalee
Project Start
Project End
Budget Start
Budget End
Support Year
26
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02115
Larman, H Benjamin; Liang, Anthony C; Elledge, Stephen J et al. (2014) Discovery of protein interactions using parallel analysis of translated ORFs (PLATO). Nat Protoc 9:90-103
Wang, Charlotte I; Alekseyenko, Artyom A; LeRoy, Gary et al. (2013) Chromatin proteins captured by ChIP-mass spectrometry are linked to dosage compensation in Drosophila. Nat Struct Mol Biol 20:202-9
Emanuele, Michael J; Elia, Andrew E H; Xu, Qikai et al. (2011) Global identification of modular cullin-RING ligase substrates. Cell 147:459-74
Shen, Xi; Do, Huong; Li, Yongjiang et al. (2009) Recruitment of fanconi anemia and breast cancer proteins to DNA damage sites is differentially governed by replication. Mol Cell 35:716-23
Wang, Xin; Zou, Lee; Lu, Tao et al. (2006) Rad17 phosphorylation is required for claspin recruitment and Chk1 activation in response to replication stress. Mol Cell 23:331-41
Li, Ju-mei; Li, Yumei; Elledge, Stephen J (2005) Genetic analysis of the kinetochore DASH complex reveals an antagonistic relationship with the ras/protein kinase A pathway and a novel subunit required for Ask1 association. Mol Cell Biol 25:767-78
Lin, Shiaw-Yih; Li, Kaiyi; Stewart, Grant S et al. (2004) Human Claspin works with BRCA1 to both positively and negatively regulate cell proliferation. Proc Natl Acad Sci U S A 101:6484-9
Wang, Yanchang; Shirogane, Takahiro; Liu, Dou et al. (2003) Exit from exit: resetting the cell cycle through Amn1 inhibition of G protein signaling. Cell 112:697-709
Zou, Lee; Liu, Dou; Elledge, Stephen J (2003) Replication protein A-mediated recruitment and activation of Rad17 complexes. Proc Natl Acad Sci U S A 100:13827-32
Osborn, Alexander J; Elledge, Stephen J (2003) Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53. Genes Dev 17:1755-67

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