Cells continuously experience DNA damage caused by replication errors, metabolically induced oxidative damage, and exogenous mutagens. DNA damage triggers orchestrated cellular responses that include mobilization of repair machinery and activation of cell cycle checkpoints, which arrest cells in G1 and G2/M and slow progression through S phase. Genetic and pharmacologic data demonstrate that in organisms as diverse as yeast and humans, disruption of checkpoint function leads to genetic instability, which correlates with acquisition of oncogenic mutations in mammals. Genetic studies in yeast show that inactivation of checkpoint genes prevents DNA damage-induced cell cycle arrest and sensitizes the yeast to genotoxins. Although many of the yeast checkpoint genes have been identified, our understanding of DNA damage-induced checkpoint activation in mammals has lagged behind. To identify potential regulators of the mammalian DNA- damage response, we cloned hRad1, hRad9, and hHus1, which are human homologs of the S. pombe checkpoint genes, rad1, rad9, and hus1, and we initiated a study to examine the biochemical and cellular functions of these proteins in mammals. Our results demonstrate that these proteins form a complex in human cells. Two members of the complex, hRad9 and hRad1, are phosphorylated and associated with chromatin in response to DNA damage, thus demonstrating that these proteins form a damage-responsive checkpoint complex. Moreover, we provide new data demonstrating that DNA damage provokes a redistribution of hRad9 into nuclear foci. We now propose to extend these findings and define the biochemical functions of hRad9 and identify its roles in cellular checkpoint responses.
The Specific Aims of the project are to: 1) determine whether nuclear retention of hRad9 after DNA damage reflects a biochemical sensing mechanism for damaged DNA; 2) analyze the role of hRad9 in checkpoint signaling activation and cell cycle arrest following DNA damage; 3) identify the basal and DNA damage-induced hRad9 phosphorylation sites and determine what role(s) they play in mediating interactions with the hRad1/hHus1 1 heterodimer and in checkpoint activation. Collectively, these studies will reveal novel insights into the pathways that recognize DNA damage and activate intracellular signaling pathways that regulate cell cycle arrest and maintain genome integrity. Additionally, understanding the molecular details of this pathway will identify novel targets to sensitize cells to therapeutic DNA-damaging agents, such as ionizing radiation and anti-tumor chemotherapeutics.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA084321-02
Application #
6377689
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Pelroy, Richard
Project Start
2000-07-01
Project End
2004-06-30
Budget Start
2001-07-01
Budget End
2002-06-30
Support Year
2
Fiscal Year
2001
Total Cost
$222,233
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
City
Rochester
State
MN
Country
United States
Zip Code
55905
Karnitz, Larry M; Zou, Lee (2015) Molecular Pathways: Targeting ATR in Cancer Therapy. Clin Cancer Res 21:4780-5
Schenk, Erin L; Koh, Brian D; Flatten, Karen S et al. (2012) Effects of selective checkpoint kinase 1 inhibition on cytarabine cytotoxicity in acute myelogenous leukemia cells in vitro. Clin Cancer Res 18:5364-73
Huehls, Amelia M; Wagner, Jill M; Huntoon, Catherine J et al. (2012) Identification of DNA repair pathways that affect the survival of ovarian cancer cells treated with a poly(ADP-ribose) polymerase inhibitor in a novel drug combination. Mol Pharmacol 82:767-76
Huehls, Amelia M; Wagner, Jill M; Huntoon, Catherine J et al. (2011) Poly(ADP-Ribose) polymerase inhibition synergizes with 5-fluorodeoxyuridine but not 5-fluorouracil in ovarian cancer cells. Cancer Res 71:4944-54
Geng, Liyi; Huehls, Amelia M; Wagner, Jill M et al. (2011) Checkpoint signaling, base excision repair, and PARP promote survival of colon cancer cells treated with 5-fluorodeoxyuridine but not 5-fluorouracil. PLoS One 6:e28862
Wagner, Jill M; Karnitz, Larry M (2009) Cisplatin-induced DNA damage activates replication checkpoint signaling components that differentially affect tumor cell survival. Mol Pharmacol 76:208-14
Leonard, Jennifer M; Ye, Hong; Wetmore, Cynthia et al. (2008) Sonic Hedgehog signaling impairs ionizing radiation-induced checkpoint activation and induces genomic instability. J Cell Biol 183:385-91
Delacroix, Sinny; Wagner, Jill M; Kobayashi, Masahiko et al. (2007) The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1. Genes Dev 21:1472-7
Karnitz, Larry M; Flatten, Karen S; Wagner, Jill M et al. (2005) Gemcitabine-induced activation of checkpoint signaling pathways that affect tumor cell survival. Mol Pharmacol 68:1636-44
Hackbarth, Jennifer S; Lee, Sun-Hee; Meng, Xue Wei et al. (2004) S-peptide epitope tagging for protein purification, expression monitoring, and localization in mammalian cells. Biotechniques 37:835-9

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