Human 53BP1 (p53 tumor suppresor binding protein 1) has BRCT domains with high homology to the S. cerevisiae Rad9 (Sc_Rad9) and S. pombe Rhp9/Crb2 (Sp_Rhp9/Crb2) DNA damage checkpoint proteins. To examine whether 53BP1 has a role in the cellular response to DNA damage, we probed its intracellular localization by immunofluorescence. In untreated primary cells and U2OS osteosarcoma cells, 53BP1 exhibited diffuse nuclear staining; but localized to discreet nuclear foci rapidly after exposure of the cells to ionizing radiation (IR). Several lines of evidence suggest that these foci are sites of DNA double-strand breaks (DSBs). Based on these preliminary results and the sequence homology between 53BP1 and the yeast DNA damage checkpoint proteins Sc_Rad9 and Sp_Rhp9/Crb2, we hypothesize that 53BP1 is an early participant in the cellular response to DNA DSBs and has a role in the DNA damage checkpoint response. To test these hypotheses, we propose the following Specific Aims: I. Elucidate the mechanism by which 53BP1 is targeted to JR-induced foci. We will map the domain that targets 53BP1 to JR-induced foci and explore the mechanism by which this domain is targeted to these foci, including examining whether this domain binds to DNA or chromatin. 2. Determine whether 53BP1 functions as a DNA damage checkpoint. We will study the DNA damage checkpoint response (cell cycle arrest, apoptosis, radiosensitivity, activation of ATM, ATR, Chk1, Chk2, p53 and Cdc25C) in cells in which 53BP1 function is upregulated or downregulated. We will also study the physical interaction of 53BP1 and p53 and its significance for activation of p53 in response to DNA damage. 3. Unlike normal cells, which have 53BP1 foci only in response to IR, many human cancer cell lines show 53BP1 foci even in the absence of exogenous DNA damaging agents. We will characterize the 53BP1 foci in non-irradiated cancer cells to determine, if they represent sites of DNA DSBs. We will also examine whether we can exploit the difference in 53BP1 behavior between normal and cancer cells to specifically target tumor cells. Overall, this proposal aims to contribute to our understanding of DNA damage checkpoints and to characterize a novel, potentially significant, difference between normal and cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA076367-06
Application #
6607622
Study Section
Pathology B Study Section (PTHB)
Program Officer
Pelroy, Richard
Project Start
1998-08-10
Project End
2006-07-31
Budget Start
2003-08-01
Budget End
2004-07-31
Support Year
6
Fiscal Year
2003
Total Cost
$281,530
Indirect Cost
Name
Wistar Institute
Department
Type
DUNS #
075524595
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Mochan, Tamara A; Venere, Monica; DiTullio Jr, Richard A et al. (2004) 53BP1, an activator of ATM in response to DNA damage. DNA Repair (Amst) 3:945-52
Huyen, Yentram; Jeffrey, Philip D; Derry, W Brent et al. (2004) Structural differences in the DNA binding domains of human p53 and its C. elegans ortholog Cep-1. Structure 12:1237-43
Halazonetis, Thanos D (2004) Constitutively active DNA damage checkpoint pathways as the driving force for the high frequency of p53 mutations in human cancer. DNA Repair (Amst) 3:1057-62
DiTullio Jr, Richard A; Mochan, Tamara A; Venere, Monica et al. (2002) 53BP1 functions in an ATM-dependent checkpoint pathway that is constitutively activated in human cancer. Nat Cell Biol 4:998-1002
Stavridi, E S; Chehab, N H; Malikzay, A et al. (2001) Substitutions that compromise the ionizing radiation-induced association of p53 with 14-3-3 proteins also compromise the ability of p53 to induce cell cycle arrest. Cancer Res 61:7030-3
Chehab, N H; Malikzay, A; Appel, M et al. (2000) Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53. Genes Dev 14:278-88
Schultz, L B; Chehab, N H; Malikzay, A et al. (2000) The DNA damage checkpoint and human cancer. Cold Spring Harb Symp Quant Biol 65:489-98
Schultz, L B; Chehab, N H; Malikzay, A et al. (2000) p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks. J Cell Biol 151:1381-90
Chehab, N H; Malikzay, A; Stavridi, E S et al. (1999) Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage. Proc Natl Acad Sci U S A 96:13777-82