DNA double-strand breaks (DSBs) arise from normal cellular processes and from exogenous sources, such as ionizing radiation. Cells have multiple mechanisms to repair DSBs, including nonhomologous end-joining (NHEJ) and homologous recombination (HR). Lack of repair can lead to genetic loss and cell death, whereas misrepair of DSBs can lead to genomic rearrangements such as chromosomal translocations that are associated with tumorigenesis. Sequencing of breakpoint junctions from patients indicates that translocations primarily arise by some type of NHEJ. A relatively well-characterized "canonical" NHEJ pathway has been defined, but NHEJ can also occur without canonical NHEJ components, and is loosely termed alternative NHEJ (alt-NHEJ).
The specific aims are: 1. To further elucidate mechanisms and the role of alt-NHEJ in mouse cells. Alt-NHEJ is reported to be the primary mechanism for chromosomal translocation formation in mouse cells. We will determine what factors promote and suppress translocations and alt-NHEJ, with a focus on factors that are implicated in controlling DNA end resection. The development of an end resection assay is also proposed. Further, the role of the major alt-NHEJ DNA ligase, Lig3, will be examined in vivo. 2. To develop a clinically relevant translocation system in human stem cells. Specifically, we will induce the Ewing sarcoma EWS-FLI1 translocation in genetically unmodified mesenchymal precursor and stem cells and determine its acute consequence on cell proliferation. Intra-locus deletions within the FLI1 locus will also be interrogated. Both typs of genome destabilizing repair involving 2 DSBs (translocations, deletions) will be investigated relative to genome stabilizing repair of a single DSB. 3. To elucidate mechanisms of chromosomal translocation formation and intra-locus deletions in human cell mutants. Human cells have been reported to have higher canonical NHEJ activity than rodent cells. A key question will be how canonical and alt-NHEJ participate in translocation formation, determined using human cell mutants in both pathways, with a focus on DNA ligase mutants. Intra-locus deletions and single-break DSB repair will also be examined, providing a comprehensive analysis of chromosomal DSB repair in human cells and the genetic requirements. The effect of Lig3 loss on the radiation response will also be assessed.

Public Health Relevance

DNA double-strand breaks compromise the integrity of the genome and so must be repaired. However, misrepair can lead to genomic rearrangements, including translocations deletions, which are associated with many tumor types. This project will address fundamental questions about the mechanisms of chromosomal translocation formation and other types of break repair in mouse and human cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM054668-13A1
Application #
8584903
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Janes, Daniel E
Project Start
1998-01-01
Project End
2017-05-31
Budget Start
2013-09-01
Budget End
2014-05-31
Support Year
13
Fiscal Year
2013
Total Cost
$446,966
Indirect Cost
$194,157
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
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
Ghezraoui, Hind; Piganeau, Marion; Renouf, Benjamin et al. (2014) Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. Mol Cell 55:829-42
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 69:171-8
Piganeau, Marion; Ghezraoui, Hind; De Cian, Anne et al. (2013) Cancer translocations in human cells induced by zinc finger and TALE nucleases. Genome Res 23:1182-93
Jasin, Maria; Rothstein, Rodney (2013) Repair of strand breaks by homologous recombination. Cold Spring Harb Perspect Biol 5:a012740
Kass, Elizabeth M; Helgadottir, Hildur R; Chen, Chun-Chin et al. (2013) Double-strand break repair by homologous recombination in primary mouse somatic cells requires BRCA1 but not the ATM kinase. Proc Natl Acad Sci U S A 110:5564-9
Nakanishi, Koji; Cavallo, Francesca; Perrouault, Loic et al. (2011) Homology-directed Fanconi anemia pathway cross-link repair is dependent on DNA replication. Nat Struct Mol Biol 18:500-3
Siaud, Nicolas; Barbera, Maria A; Egashira, Akinori et al. (2011) Plasticity of BRCA2 function in homologous recombination: genetic interactions of the PALB2 and DNA binding domains. PLoS Genet 7:e1002409
Simsek, Deniz; Brunet, Erika; Wong, Sunnie Yan-Wai et al. (2011) DNA ligase III promotes alternative nonhomologous end-joining during chromosomal translocation formation. PLoS Genet 7:e1002080
Zhang, Yu; Jasin, Maria (2011) An essential role for CtIP in chromosomal translocation formation through an alternative end-joining pathway. Nat Struct Mol Biol 18:80-4

Showing the most recent 10 out of 28 publications