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.
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.
|Alotaibi, Moureq; Sharma, Khushboo; Saleh, Tareq et al. (2016) Radiosensitization by PARP Inhibition in DNA Repair Proficient and Deficient Tumor Cells: Proliferative Recovery in Senescent Cells. Radiat Res 185:229-45|
|Batenburg, Nicole L; Thompson, Elizabeth L; Hendrickson, Eric A et al. (2015) Cockayne syndrome group B protein regulates DNA double-strand break repair and checkpoint activation. EMBO J 34:1399-416|
|Castella, Maria; Jacquemont, Celine; Thompson, Elizabeth L et al. (2015) FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2. PLoS Genet 11:e1005563|
|Roy, Sunetra; de Melo, Abinadabe J; Xu, Yao et al. (2015) XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation. Mol Cell Biol 35:3017-28|
|Napier, Christine E; Huschtscha, Lily I; Harvey, Adam et al. (2015) ATRX represses alternative lengthening of telomeres. Oncotarget 6:16543-58|
|Jones, Rhiannon E; Oh, Sehyun; Grimstead, Julia W et al. (2014) Escape from telomere-driven crisis is DNA ligase III dependent. Cell Rep 8:1063-76|
|Kan, Yinan; Ruis, Brian; Lin, Sherry et al. (2014) The mechanism of gene targeting in human somatic cells. PLoS Genet 10:e1004251|
|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; Harvey, Adam; Zimbric, Jacob et al. (2014) DNA ligase III and DNA ligase IV carry out genetically distinct forms of end joining in human somatic cells. DNA Repair (Amst) 21:97-110|
|Karanja, Kenneth K; Lee, Eu Han; Hendrickson, Eric A et al. (2014) Preventing over-resection by DNA2 helicase/nuclease suppresses repair defects in Fanconi anemia cells. Cell Cycle 13:1540-50|
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