The long term goal of this proposal is to understand the molecular mechanisms that repair DNA damage induced by DNA topoisomerase I (Top1) -targeting anticancer drugs such as camptothecin (CPT) and its analogues. These studies will identify novel mechanisms by which cells integrate DNA repair with cell cycle progression to maintain genome stability. Top1 plays an important role in maintenance of genome integrity by resolving topological strain during vital cellular processes such as DNA replication, transcription and chromatin remodeling. Covalently trapping of Top1-DNA covalent complexes by CPT induces replication and transcription mediated lethal DNA lesions (such as DSBs). However, mechanisms underlying repair of these lesions are not well understood. Preliminary studies reported here, describe a novel connection between DNA repair protein Rad18 and Fanconi Anemia (FA) pathway in response to CPT induced DNA damage. The Rad18 deficient cells or cells expressing mutant Rad18 defective in E3 ligase activity, fail to efficiently recruit FANCD2 (a key component of FA pathway activation) to chromatin making cells hypersensitive to CPT. Many studies also suggest that Rad18 plays an important role in repair of CPT induced DNA damage in different phases of the cell cycle. However, the molecular networks that orchestrate these pathways with specific stages of the cell cycle are poorly understood.
The specific aims i n this proposal are designed to understand the roles of Rad18 in Top1 inhibitors induced DNA damage and its regulation of different DNA repair pathways. Studies proposed under Specific Aim1 will focus on determining mechanisms by which Rad18 activates FA pathway in response to CPT, using a variety of biochemical and genetic tools.
The Specific Aim 2 will test the hypothesis that Rad18 and FA pathway components affect cells'recovery from CPT induced DNA damage. The roles that Rad18 and FA pathway play in regulation of S-phase checkpoint, fork stability, and the initiation and elongation events of DNA replication will also be determined. Some cutting edge techniques such as protein-DNA labeling and molecular combing will be used to test this hypothesis. Experiments under Specific Aim 3 will determine the roles of Rad18 in different phases of the cell cycle in response to CPT poisoning of Top1.Outcomes from this study will significantly contribute to understanding the mechanisms by which cells maintain genome stability. Proposed investigations into Rad18's interaction with FA pathway and its regulation during cell cycle will potentially reveal new molecular targets and thereby, novel therapeutic combinations for enhancing targeted destruction of cancer cells by Top1 inhibitors.

Public Health Relevance

The long term goal of this proposal is to understand the molecular mechanisms that repair DNA damage induced by anticancer drug camptothecin and its analogues topotecan and irinotecan. The proposed studies may identify novel mechanisms by which cells integrate DNA repair with cell cycle progression to maintaining genome integrity and potential new targets for augmenting killing of cancer cells by these drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098956-04
Application #
8598907
Study Section
Basic Mechanisms of Cancer Therapeutics Study Section (BMCT)
Program Officer
Janes, Daniel E
Project Start
2012-04-01
Project End
2016-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
4
Fiscal Year
2014
Total Cost
$205,821
Indirect Cost
$67,221
Name
University of South Alabama
Department
Type
Schools of Medicine
DUNS #
172750234
City
Mobile
State
AL
Country
United States
Zip Code
36688
Tripathi, Kaushlendra; Mani, Chinnadurai; Clark, David W et al. (2016) Rad18 is required for functional interactions between FANCD2, BRCA2, and Rad51 to repair DNA topoisomerase 1-poisons induced lesions and promote fork recovery. Oncotarget 7:12537-53
Somasagara, Ranganatha R; Tripathi, Kaushlendra; Spencer, Sebastian M et al. (2016) Rad6 upregulation promotes stem cell-like characteristics and platinum resistance in ovarian cancer. Biochem Biophys Res Commun 469:449-55
Kothayer, Hend; Spencer, Sebastian M; Tripathi, Kaushlendra et al. (2016) Synthesis and in vitro anticancer evaluation of some 4,6-diamino-1,3,5-triazine-2-carbohydrazides as Rad6 ubiquitin conjugating enzyme inhibitors. Bioorg Med Chem Lett 26:2030-4
Tripathi, Kaushlendra; Hussein, Usama K; Anupalli, Roja et al. (2015) Allyl isothiocyanate induces replication-associated DNA damage response in NSCLC cells and sensitizes to ionizing radiation. Oncotarget 6:5237-52
Clark, David W; Tripathi, Kaushlendra; Dorsman, Josephine C et al. (2015) FANCJ protein is important for the stability of FANCD2/FANCI proteins and protects them from proteasome and caspase-3 dependent degradation. Oncotarget 6:28816-32
Palle, Komaraiah; Mani, Chinnadurai; Tripathi, Kaushlendra et al. (2015) Aberrant GLI1 Activation in DNA Damage Response, Carcinogenesis and Chemoresistance. Cancers (Basel) 7:2330-51
Meng, Erhong; Mitra, Aparna; Tripathi, Kaushlendra et al. (2014) ALDH1A1 maintains ovarian cancer stem cell-like properties by altered regulation of cell cycle checkpoint and DNA repair network signaling. PLoS One 9:e107142
Tripathi, Kaushlendra; Mani, Chinnadurai; Barnett, Reagan et al. (2014) Gli1 protein regulates the S-phase checkpoint in tumor cells via Bid protein, and its inhibition sensitizes to DNA topoisomerase 1 inhibitors. J Biol Chem 289:31513-25