Transient arrest of the eukaryotic cell cycle is an actively regulated response to DNA-damaging agents and other agents perturbing normal cell cycle progression, such as replication inhibitors or poisons of the mitotic spindle. Such checkpoints are quite frequently compromised in tumor cells and checkpoint failure may constitute an important factor contributing to the genetic instability that is commonly associated with the establishment of malignancy. Differential checkpoint responses in tumor cells vs. wild type cells may also translate into differential drug sensitivities since the majority of effective anticancer drugs are DNA-damaging agents or other agents that will induce a checkpoint arrest response. Rad17 of budding yeast Saccharomyces cerevisiae is an evolutionary conserved protein with an indispensable role in checkpoint arrest in G1 and G2 in response to DNA damage. Analysis of Rad17 in the yeast-two hybrid system suggested an increased homomeric complex formation in the presence of DNA damage. Such an effect can be demonstrated in a technically simple gradient assay performed on plates (DIPI assay, for DNA- damage-induced protein interaction). It is proposed to explore this system further with the dual goal of providing a screening system for high-throughput profiling of the checkpoint-activating or -modifying potential of candidate anticancer agents and at the same time elucidating the molecular basis of the Rad17 protein-protein interactions. We intend... To confirm a stimulation of Rad17/Rad17 complex formation by DNA damage through biochemical analysis. To identify regions of the Rad17 protein that are required for self- interaction and its regulation by DNA damage and to analyze the phenotype conferred by alterations in such regions. To develop the gradient assay (""""""""DIPI assay"""""""") into a useful profiling tool by investigating the outcome for various substances, by comparing the assay to an established test for genotoxicity and by determining the influence of different mutant backgrounds.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA087381-05
Application #
6615133
Study Section
Special Emphasis Panel (ZCA1-SRRB-E (M1))
Program Officer
Forry, Suzanne L
Project Start
2000-08-01
Project End
2005-07-31
Budget Start
2003-08-01
Budget End
2004-07-31
Support Year
5
Fiscal Year
2003
Total Cost
$212,856
Indirect Cost
Name
University of North Texas
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
110091808
City
Fort Worth
State
TX
Country
United States
Zip Code
76107
Jain, Dilip; Patel, Nila; Shelton, Melanie et al. (2010) Enhancement of cisplatin sensitivity by NSC109268 in budding yeast and human cancer cells is associated with inhibition of S-phase progression. Cancer Chemother Pharmacol 66:945-52
Pabla, Ritu; Pawar, Vaibhav; Zhang, Hong et al. (2006) Characterization of checkpoint responses to DNA damage in Saccharomyces cerevisiae: basic protocols. Methods Enzymol 409:101-17
Giannattasio, Michele; Lazzaro, Federico; Siede, Wolfram et al. (2004) DNA decay and limited Rad53 activation after liquid holding of UV-treated nucleotide excision repair deficient S. cerevisiae cells. DNA Repair (Amst) 3:1591-9
Zhang, Hong; Siede, Wolfram (2004) Analysis of the budding yeast Saccharomyces cerevisiae cell cycle by morphological criteria and flow cytometry. Methods Mol Biol 241:77-91
Evert, Barbara A; Salmon, Tiffany B; Song, Binwei et al. (2004) Spontaneous DNA damage in Saccharomyces cerevisiae elicits phenotypic properties similar to cancer cells. J Biol Chem 279:22585-94
Zhang, Hong; Taylor, Jena; Siede, Wolfram (2003) Checkpoint arrest signaling in response to UV damage is independent of nucleotide excision repair in Saccharomyces cerevisiae. J Biol Chem 278:9382-7
Zhang, Hong; Siede, Wolfram (2003) Validation of a novel assay for checkpoint responses: characterization of camptothecin derivatives in Saccharomyces cerevisiae. Mutat Res 527:37-48
Zhang, Hong; Siede, Wolfram (2002) UV-induced T-->C transition at a TT photoproduct site is dependent on Saccharomyces cerevisiae polymerase eta in vivo. Nucleic Acids Res 30:1262-7
Zhang, H; Zhu, Z; Vidanes, G et al. (2001) Characterization of DNA damage-stimulated self-interaction of Saccharomyces cerevisiae checkpoint protein Rad17p. J Biol Chem 276:26715-23