Ionizing radiation and other DNA damaging agents can break chromosomes. Mammalian cells are able to repair chromosomal breaks, including double-strand breaks (DSBs), although the mechanisms of the repair processes are insufficiently understood. Recent developments have led to the identification of gene products defective in ionizing radiation sensitive cell lines and to the cloning of mammalian homologs to yeast genes involved in recombinational repair of DSBs. In the past two years, this lab has demonstrated that chromosomal DSBs in mammalian cells are repaired by recombinational repair, as well as by nonhomologous mechanisms. For these studies, they have developed expression systems for a rare-cutting, site specific endonuclease in order to introduce chromosomal DSBs at repair substrates integrated into the genome. The researchers plan to exploit this system to define the pathways of recombinational repair of chromosomal DSBs in mammalian cells. They hypothesize that recombinational repair pathways will be conserved between yeast and mammalian cells. Thus, they propose to determine if recombination occurs by a conservative pathway, resulting in both crossover and noncrossover products, and if nonconservative recombination occurs, as well as if homologous crossovers occur between sister chromatids and within chromatids. They also plan to determine the effects of heterology and cell cycle stage on recombination, since two major sources of homology for repair of DSBs in mammalian genomes are divergent repetitive elements and sister chromatids. A set of cell mutants will also be screened for defects in recombinational repair. The long-term goals are to understand in detail how mammalian cells repair a major assault on their genetic integrity: the DNA DSB. Radiation can result in chromosome abnormalities and disease states. Yet, it is also a therapy in the treatments of cancers. The exchange of information between related DNA sequences (homologous recombination), apparently promoted by DSBs, is a key process leading to genetic diversity in all organisms that have been examined and plays a critical role in proper chromosome segregation at meiosis. Understanding homologous recombination and the repair of DSBs in mammalian cells, therefore, is fundamental to understanding the maintenance of integrity of the eukaryotic genome. The understanding that is gained has a long-range impact on health, medicine, and basic science.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM054668-01A1
Application #
2471331
Study Section
Radiation Study Section (RAD)
Project Start
1998-01-01
Project End
2001-12-31
Budget Start
1998-01-01
Budget End
1998-12-31
Support Year
1
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
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Browning, Cynthia L; Qin, Qin; Kelly, Deborah F et al. (2016) Prolonged Particulate Hexavalent Chromium Exposure Suppresses Homologous Recombination Repair in Human Lung Cells. Toxicol Sci 153:70-8
Jasin, Maria; Haber, James E (2016) The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair. DNA Repair (Amst) 44:6-16
Prakash, Rohit; Zhang, Yu; Feng, Weiran et al. (2015) Homologous recombination and human health: the roles of BRCA1, BRCA2, and associated proteins. Cold Spring Harb Perspect Biol 7:a016600
Goglia, Alexander G; Delsite, Robert; Luz, Antonio N et al. (2015) Identification of novel radiosensitizers in a high-throughput, cell-based screen for DSB repair inhibitors. Mol Cancer Ther 14:326-42
Vriend, Lianne E M; Jasin, Maria; Krawczyk, Przemek M (2014) Assaying break and nick-induced homologous recombination in mammalian cells using the DR-GFP reporter and Cas9 nucleases. Methods Enzymol 546:175-91
Renouf, Benjamin; Piganeau, Marion; Ghezraoui, Hind et al. (2014) Creating cancer translocations in human cells using Cas9 DSBs and nCas9 paired nicks. Methods Enzymol 546:251-71
Zhang, Yu; Vanoli, Fabio; LaRocque, Jeannine R et al. (2014) Biallelic targeting of expressed genes in mouse embryonic stem cells using the Cas9 system. Methods 69:171-178
Ghezraoui, Hind; Piganeau, Marion; Renouf, Benjamin et al. (2014) Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. Mol Cell 55:829-842
Jasin, Maria; Rothstein, Rodney (2013) Repair of strand breaks by homologous recombination. Cold Spring Harb Perspect Biol 5:a012740

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