Our long-term research goal is to understand the molecular mechanisms of the cellular response to DNA double-strand breaks (DSB), an anti-cancer system crucial for the maintenance of genome stability. This proposal focuses specifically on the mechanisms of DSB resection, a multi-step process required for both activation of the DNA damage checkpoint and initiation of homology-based DNA repair. DSB resection occurs via nucleolytic removal of 5'-strand DNA from the DNA end. The resulting 3'ssDNA structure serves as the signal for activation of the ATR checkpoint and as a key intermediate for homologous recombination. In vertebrates, several proteins including the Mre11-Rad50-NBS1 (MRN) complex, CtIP, Exo1, DNA2 and BLM have been shown to be involved in DSB resection. Mutation or deregulation of the genes encoding these proteins is associated with genome instability and predisposition to cancer. However, despite its critical importance in genome maintenance and disease prevention, the molecular mechanisms and regulation of the DNA end resection process remain poorly understood. Building upon our recently published work and our extensive new data, in this proposal we described a series of innovative, hypothesis-driven studies to elucidate three key aspects of the DSB resection process. (1) We will determine how DSB resection is initiated and define the role of WRN, BLM, MRN and CtIP in the initiation stage of resection. (2) We will define how the initial stage of DNA resection, which is MRN/CtIP-dependent, is switched to the extended resection phase that is dependent on Exo1 and Dna2. (3) We will delineate the interplay between DNA end resection and the checkpoint pathway and determine how this interplay controls the termination of DSB resection. To achieve these goals, we will carry out both in vitro and in vivo studies using a unique combination of experimental systems and tools, including Xenopus egg extracts, cultured human cells and a laser micro-irradiation technique to create DSBs in cells. This work will significantly advance the field by identifying new components and acquiring in-depth mechanistic understanding of the DSB resection process in the context of the overall DNA damage response. As an Early-Stage Investigator at Washington University School of Medicine, I will take advantage of my group's strengths in biochemistry and cell biology to decipher the DNA damage response network, and collaborate with other groups to translate our findings into better understanding and treatment of cancer.

Public Health Relevance

Our cells are constantly at risk for DNA double-strand break damage that, if not repaired, can cause or contribute to human diseases such as cancer, premature aging, immunodeficiency and developmental disorders. The DNA damage checkpoint and repair pathways are major defense systems that combat DNA lesions in cells and prevent the formation and development of cancer. This grant proposal is designed to delineate the mechanism of DNA double-strand break processing that is essential for activating the DNA damage checkpoint and initiating a major DNA repair pathway-with the long-term goal of improving the efficiency of breast cancer treatment.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098535-02
Application #
8453376
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Janes, Daniel E
Project Start
2012-04-05
Project End
2016-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
2
Fiscal Year
2013
Total Cost
$278,692
Indirect Cost
$95,342
Name
Washington University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Nickless, Andrew; Jackson, Erin; Marasa, Jayne et al. (2014) Intracellular calcium regulates nonsense-mediated mRNA decay. Nat Med 20:961-6
Chen, Xiaoqing; Paudyal, Sharad C; Chin, Re-I et al. (2013) PCNA promotes processive DNA end resection by Exo1. Nucleic Acids Res 41:9325-38
Liu, Chao; Wu, Jiaxue; Paudyal, Sharad C et al. (2013) CHFR is important for the first wave of ubiquitination at DNA damage sites. Nucleic Acids Res 41:1698-710