Abstract

DNA damage response networks are common targets for carcinogenic mutations and may be excellent targets for response-modifying drugs. The central core of these networks consists of phosphatidyl inositol 3-kinase-related kinases (PIKKs), e.g. human Atm and Atr, which activate downstream pathways in response to DNA damage. PIKKs phosphorylate components of complexes that are recruited to damageinduced foci, including the Mrell/Rad50/Nbsl complex, 53BP1, and Brcal, and activate effector kinases Chkl and Chk2 that amplify the signal and help convey it to effector proteins. In budding yeast, the PIKK Mecl (Atm/Atr ortholog) is coupled to protein kinase Rad53 (Chk2 ortholog) through Mecl-dependent phosphorylation of the """"""""mediator"""""""" Rad9 (unrelated to human Rad9). Mammalian BRCA1, 53BP1, and now MDC1/NFBD1 are candidate Rad9 orthologs that resemble Rad9 in having twin carboxyl terminal BRCT domains. Like Rad9, they undergo PIKK-dependent phosphorylation, form damage-induced complexes with other checkpoint proteins and are recruited to sites of DNA damage. MDC1/NFBD1 is intimately involved in DNA damage response mechanisms. The PIKK-Mediator-Chk core of DNA damage signaling will be investigated using both the simpler yeast cascade Mecl to Rad9 to Rad53, and the more complex interactions of ATM/ATR in regulation of NFBD1. ? ? Aim 1. A combination of biochemical and molecular methods will be used to determine the mechanism ? through which the mediator Rad9 and the PIKK Mecl regulate Rad53. ? ? Aim 2.. siRNA oligonucleotide knockdowns will be used to identify unique and overlapping functions of BRCA1, 53BP1, and NFBD1 in DNA checkpoint responses. ? ? Aim 3. Molecular techniques will be used for functional analysis of NFBD1/MDC1 in the activities ? identified in Aim 2. ? ?

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA082257-08
Application #
7230468
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Pelroy, Richard
Project Start
1999-07-01
Project End
2010-04-30
Budget Start
2007-05-01
Budget End
2008-04-30
Support Year
8
Fiscal Year
2007
Total Cost
$263,251
Indirect Cost

  Institution

Name
Yale University
Department
Pathology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520

  Publications

Liu, Jinping; Luo, Shukun; Zhao, Hongchang et al. (2012) Structural mechanism of the phosphorylation-dependent dimerization of the MDC1 forkhead-associated domain. Nucleic Acids Res 40:3898-912
Wilson, Kathleen A; Colavito, Sierra A; Schulz, Vincent et al. (2011) NFBD1/MDC1 regulates Cav1 and Cav2 independently of DNA damage and p53. Mol Cancer Res 9:766-81
Mok, Janine; Kim, Philip M; Lam, Hugo Y K et al. (2010) Deciphering protein kinase specificity through large-scale analysis of yeast phosphorylation site motifs. Sci Signal 3:ra12
Golan, Amnon; Pick, Elah; Tsvetkov, Lyuben et al. (2010) Centrosomal Chk2 in DNA damage responses and cell cycle progression. Cell Cycle 9:2647-56
Stern, David F (2009) BRCTing up is hard to do. Mol Cell 33:137-8
Wilson, Kathleen A; Stern, David F (2008) NFBD1/MDC1, 53BP1 and BRCA1 have both redundant and unique roles in the ATM pathway. Cell Cycle 7:3584-94
Jia-Lin Ma, Nianhan; Stern, David F (2008) Regulation of the Rad53 protein kinase in signal amplification by oligomer assembly and disassembly. Cell Cycle 7:808-17
Ma, Jia-Lin; Lee, Soo-Jung; Duong, Jimmy K et al. (2006) Activation of the checkpoint kinase Rad53 by the phosphatidyl inositol kinase-like kinase Mec1. J Biol Chem 281:3954-63
Li, Jia; Young, C S H; Lizardi, Paul M et al. (2005) In situ detection of specific DNA double strand breaks using rolling circle amplification. Cell Cycle 4:1767-73
Li, Jia; Stern, David F (2005) Regulation of CHK2 by DNA-dependent protein kinase. J Biol Chem 280:12041-50

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