We propose to study the impact of loss-of-function mutations of DNA DSB (double-strand break) repair genes on end joining processes in human cells. We will focus heavily, but not exclusively, on the heterodimeric (Ku70 and Ku86 subunits) Ku DNA end-binding complex and LIGIII (DNA ligase III). Ku has a storied history and it has been known for decades that it is required for most of the generalized DSB repair occurring in somatic cells as well as being critical for the proper development of the vertebrate immune system. Ku performs these duties as part of a repair pathway referred to as C-NHEJ (classic-non-homologous end joining). In work published this year, we demonstrated that Ku has a hitherto underappreciated function as a suppressor of the other competing DSB repair pathways, namely A-NHEJ (alternative-NHEJ) and HR (homologous recombination). Thus, when the levels of Ku are reduced, A-NHEJ and HR, which normally comprise only a small fraction of the end joining activity in human cells, become more active. This observation has potential clinical implications. Thus, the vast majority of human cancer patients are treated either with radio- or chemotherapy (or both) and the efficacy of these anti-tumor modalities resides in their ability to generate DNA DSBs. Thus, strategies designed to increase the efficacy of tumor cell killing by radio- or chemotherapy that involve reducing the levels of Ku are likely to fail due to the up-regulation of A-NHEJ and/or HR. This is a hypothesis that we will directly test in this grant application. Moreover, the molecular mechanism by which Ku mediates this suppression is unknown and we propose genetic and reverse genetic experiments to elucidate this process. Finally, we have begun to genetically tackle the A-NHEJ pathway. A number of genes have been implicated in A-NHEJ, but the important genes remain poorly defined. One of the more compelling A- NHEJ candidates is LIGIII (DNA ligase III). To experimentally test the importance of LIGIII we have constructed a viable LIGIII-null human cel line and herein propose to characterize the impact of the loss-of- function of LIGIII on A-NHEJ. In particular, one characterization involves a novel and powerful assay system by which we can measure ionizing radiation-induced gross chromosomal rearrangements in human cells lacking LIGIII (or Ku86). To our knowledge, we are one of only a few laboratories in the world utilizing genetic, loss-of-function approaches to study DNA DSB in human cells and thus we are well positioned to gain insights into the mechanism of DSB repair that cannot be obtained elsewhere.

Public Health Relevance

DNA double-strand break repair is critical for the stable maintenance of the human genome and this belief is validated by the large number of cancer predisposition syndromes, which result from mutation of genes involved in the recognition, signaling or repair of DNA double-strand breaks. In addition, the efficacy of the major cancer therapeutic modalities (radiation and chemotherapy) depends on the ability (or inability) of tumor cells to repair the resulting DNA double-stranded breaks introduced by these agents. Thus, the continued investigation of DNA double-strand break repair is clearly relevant to the mission of NIH.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA154461-03
Application #
8460914
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Pelroy, Richard
Project Start
2011-07-07
Project End
2016-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
3
Fiscal Year
2013
Total Cost
$288,679
Indirect Cost
$93,629
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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Fattah, Farjana Jahan; Kweon, Junghun; Wang, Yongbao et al. (2014) A role for XLF in DNA repair and recombination in human somatic cells. DNA Repair (Amst) 15:39-53
Kan, Yinan; Ruis, Brian; Lin, Sherry et al. (2014) The mechanism of gene targeting in human somatic cells. PLoS Genet 10:e1004251
Yeo, Jung Eun; Lee, Eu Han; Hendrickson, Eric A et al. (2014) CtIP mediates replication fork recovery in a FANCD2-regulated manner. Hum Mol Genet 23:3695-705
Smith, Stephanie; Fox, Jennifer; Mejia, Marco et al. (2014) Histone deacetylase inhibitors selectively target homology dependent DNA repair defective cells and elevate non-homologous endjoining activity. PLoS One 9:e87203
Waters, Crystal A; Strande, Natasha T; Pryor, John M et al. (2014) The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining. Nat Commun 5:4286
Oh, Sehyun; Wang, Yongbao; Zimbric, Jacob et al. (2013) Human LIGIV is synthetically lethal with the loss of Rad54B-dependent recombination and is required for certain chromosome fusion events induced by telomere dysfunction. Nucleic Acids Res 41:1734-49
Yang, Yang; Durando, Michael; Smith-Roe, Stephanie L et al. (2013) Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage. Nucleic Acids Res 41:2296-312
Strande, Natasha; Roberts, Steven A; Oh, Sehyun et al. (2012) Specificity of the dRP/AP lyase of Ku promotes nonhomologous end joining (NHEJ) fidelity at damaged ends. J Biol Chem 287:13686-93