Among all the damages to the genome, DSBs are considered one of the most deleterious to cells. They arise from both environmental agents like ionizing radiation or chemotherapeutic drugs and normal cellular processes like DNA replication, V(D)J recombination, or meiosis. If un-repaired or improperly repaired, DSBs would cause chromosome deletions or translocations, ultimately leading to premature cell death or oncogenic transformation. Mutations in many DSB repair genes, such as Werner syndrome gene and Bloom syndrome gene, BRCA1, and BRCA2, dramatically increase the risk of cancer. Clinically, eliciting DSBs by ionizing radiation and various cancer drugs is also among the most commonly used methods to treat cancer. DSBs are repaired by two general types of pathways: non-homologous end joining (NHEJ) and homology-dependent repair (HDR). Proper choice of repair pathway is critical to genome stability. The key event in the bifurcation of the two pathways is the initial processing of DNA ends. NHEJ involves limited processing, but HDR requires extensive processing to form 3'ss-tails. Recent studies from several labs, including mine, have elucidated the basic mechanisms for resection. However, many important questions are still poorly understood. Firstly, HDR and NHEJ are both active during S-G2 phases of the cell cycle, but it is unclear what factors first determine if a DSB is channeled to resection (for HDR) or to NHEJ. Secondly, ends generated by ionizing radiation and many cancer drugs often carry damaged nucleotides. These ends can still be stably bound by NHEJ factors, but repair cannot be completed or is seriously delayed. It is unclear if they are trapped or can be re-channeled to resection for HDR. Thirdly, compared to model systems, the understanding of resection in human cells is still very limited. In this application, three specific aims are proposed to address these important questions.
Specific Aim I is designed to test the hypothesis that a key factor for determining if a DSB is resected or not is the structure of ends. DNA with ends linked to a protein adduct, which are frequently induced by many cancer drugs, will be used as a model substrate to test this hypothesis. Its repair will be rigorously analyzed by biochemical reconstitution studies in Xenopus egg extracts and with purified resection proteins.
Specific Aim II is designed to test the hypothesis that ends with damaged nucleotides are bound by NHEJ factors but then re- channeled to resection for HDR by the MRE11-RAD50-NBS1 (MRN) complex. The target protein that is dislodged from ends by the MRN-mediated mechanism will be identified.
Specific Aim III is designed to test the hypothesis that the Werner syndrome protein (WRN) and the DNA2 nuclease, which are critical for resection in Xenopus egg extracts, are also important for resection in human cells. These studies will greatly increase the understanding of how DNA ends are resected and consequently how DSB repair pathways are chosen. The key proteins involved in DSB resection and end re-channeling might be developed into new targets for drugs or as biomarkers to increase the efficiency of radiation therapy and chemotherapy of cancer.

Public Health Relevance

The research on DNA double-strand break repair pathway choice and end resection as proposed in this application has two promising applications to the improvement of public health: (1) enhancing the efficacy of radiation therapy and chemotherapy of cancer;and (2) increasing the efficiency of gene targeting technology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM057962-12A1
Application #
8372567
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Janes, Daniel E
Project Start
1998-08-01
Project End
2016-05-31
Budget Start
2012-08-01
Budget End
2013-05-31
Support Year
12
Fiscal Year
2012
Total Cost
$437,561
Indirect Cost
$192,429
Name
Research Institute of Fox Chase Cancer Center
Department
Type
DUNS #
064367329
City
Philadelphia
State
PA
Country
United States
Zip Code
19111
Liao, Shuren; Guay, Catherine; Toczylowski, Thomas et al. (2012) Analysis of MRE11's function in the 5'-->3' processing of DNA double-strand breaks. Nucleic Acids Res 40:4496-506
Liao, Shuren; Toczylowski, Thomas; Yan, Hong (2011) Mechanistic analysis of Xenopus EXO1's function in 5'-strand resection at DNA double-strand breaks. Nucleic Acids Res 39:5967-77
Yan, Hong; Toczylowski, Thomas; McCane, Jill et al. (2011) Replication protein A promotes 5'-->3' end processing during homology-dependent DNA double-strand break repair. J Cell Biol 192:251-61
Liao, Shuren; Toczylowski, Thomas; Yan, Hong (2008) Identification of the Xenopus DNA2 protein as a major nuclease for the 5'->3'strand-specific processing of DNA ends. Nucleic Acids Res 36:6091-100
Liao, Shuren; Matsumoto, Yoshihiro; Yan, Hong (2007) Biochemical reconstitution of abasic DNA lesion replication in Xenopus extracts. Nucleic Acids Res 35:5422-9
Toczylowski, Thomas; Yan, Hong (2006) Mechanistic analysis of a DNA end processing pathway mediated by the Xenopus Werner syndrome protein. J Biol Chem 281:33198-205
Yan, Hong; McCane, Jill; Toczylowski, Thomas et al. (2005) Analysis of the Xenopus Werner syndrome protein in DNA double-strand break repair. J Cell Biol 171:217-27
Chen, C Y; Graham, J; Yan, H (2001) Evidence for a replication function of FFA-1, the Xenopus orthologue of Werner syndrome protein. J Cell Biol 152:985-96
Liao, S; Graham, J; Yan, H (2000) The function of Xenopus Bloom's syndrome protein homolog (xBLM) in DNA replication. Genes Dev 14:2570-5