We propose to investigate the mechanism(s) that regulate a cell's ability to escape from the crisis caused by gradual telomere shortening. In particular, we will define the roles that the i) A-NHEJ (alternative-non- homologous end joining) pathway of DNA DSB (double-strand break) repair as well as the ii) chromatin remodeling gene, ATRX (alpha thalassemia/mental retardation syndrome, X-linked), play in this process. As normal human cells age, their telomeres gradually shorten. When the telomeres shorten significantly, the cell undergoes senescence, which is a naturally-occurring, non-proliferative barrier to cancer. If, however, a cell should suffer a transforming mutation, it can by-pass senescence and continue to proliferate until its telomeres become so short that they are non-functional. The resulting lack of end protection triggers crisis, a state that is highlighted by genomic instability as chromosomes engage in breakage:fusion:bridging cycles that almost invariably result in the death of the cell. On rare occasions a cell can reestablish its telomeres and stabilize its genome. Such cells are said to be immortalized and it is likely that they are the progenitors of most human cancers. That the (dys)regulation of telomere maintenance is also associated with aging, immortalization, and tumorigenesis in other experimental systems adds confidence to the belief that these issues are conserved and important. Here, we demonstrate that the genes LIGIII (DNA ligase III) and PARP1 {poly(ADP) ribose polymerase 1} are required for human cells to survive the crisis induced by gradual telomere shortening. LIGIII and PARP1 function in the A-NHEJ branch of DNA DSB repair. We hypothesize that it is the absence of A- NHEJ that results in the death of cells undergoing crisis and we propose to i) use structure:function approaches to define the molecular interactions required for the process, ii) identify other genes involved in crisis survival using directed approaches and genome-wide screens and iii) begin to test models for how LIGIII might mechanistically control this process. In addition, we describe our preliminary data demonstrating that ATRX is a crucial regulator of ALT (alternative lengthening of telomeres) and we describe an experimental system in which we can study the genesis of ALT. In all of these approaches we utilize the strengths of the Hendrickson and Baird laboratories. The Hendrickson laboratory excels at the technology of gene targeting to study the impact of loss-of-function mutations of genes (LIGIII, PARP1 and ATRX in this instance) on telomere maintenance. The use of gene targeting provides a facile experimental system in which null, hypomorphic, and/or conditional mutations can be introduced with rapidity into human somatic cells. The Baird laboratory is the world's leader in analyzing telomere fusion events in human cells undergoing crisis. Their ability to characterize the dynamics of single telomeric ends has provided the field's deepest understanding of the mechanism of telomere fusions in human cells. In summary, our proposed studies impact on DNA repair and telomere maintenance and the importance of understanding these processes for cancer biology is clear.

Public Health Relevance

The (dys)regulation of telomeres, the terminal structures of linear chromosomes, is associated with immortalization, aging and tumorigenesis. This fact is best exemplified by the bone marrow failure and cancer predisposition syndrome, dyskeratosis congenita, where mutations in nine genes, all of which encode factors required for telomere maintenance, are known to cause the disease. Approbation of the belief that telomere maintenance is an important area of investigation was provided by the 2009 Nobel Prizes in Physiology or Medicine, which were awarded for the discovery of telomeres and telomerase.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA190492-01A1
Application #
8960088
Study Section
Cancer Genetics Study Section (CG)
Program Officer
Witkin, Keren L
Project Start
2015-08-01
Project End
2020-07-31
Budget Start
2015-08-01
Budget End
2016-07-31
Support Year
1
Fiscal Year
2015
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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Liddiard, Kate; Ruis, Brian; Takasugi, Taylor et al. (2016) Sister chromatid telomere fusions, but not NHEJ-mediated inter-chromosomal telomere fusions, occur independently of DNA ligases 3 and 4. Genome Res 26:588-600
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
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