Telomeres play an important role in protecting the ends of chromosomes and preventing chromosome fusion. We have demonstrated that the regions near telomeres are highly sensitive to DNA double-strand breaks (DSBs), in that DSBs induced with I-SceI endonuclease near telomeres are much more likely to result in large deletions, gross chromosome rearrangements (GCRs), and chromosome instability than I-SceI-induced DSBs at other locations. Importantly, the rearrangements caused by DSBs near telomeres are the same rearrangements commonly found in human cancer cells, leading us to propose that DSBs near telomeres are an important mechanism in carcinogenesis. We have also shown that the chromosome instability caused by DSBs near telomeres can be prevented by the addition of a new telomere at the site of the DSB, a process called chromosome healing. Chromosome healing is rarely observed at DSBs at other locations, and therefore we have proposed that chromosome healing is an important mechanism for preventing chromosome instability due to DSBs near telomeres. This proposal will investigate the mechanism responsible for the sensitivity of telomeric regions to DSBs and the mechanism of regulation of chromosome healing. These studies will address the hypothesis that cis-acting telomeric proteins directly inhibit DSB repair and promote chromosome healing, consistent with evidence that the telomeric protein TRF2 inhibits the ATM and MRE11 proteins involved in the cellular response to DSBs.
In Aim 1 A we will characterize the DNA repair defect in telomeric regions by determining which DNA repair proteins co-localize with the I-SceI-induced DSB. This will involve cell lines in which the location of the DSB is marked with green fluorescent protein (GFP) by inserting 256 copies of a LacO operon adjacent to the I-SceI site, and expression of LacI-GFP fusion protein, which binds the LacO operon.
In Aim 1 B we will use cell clones containing a GFP gene and an I-SceI site adjacent to a telomere to monitor how knockdown of telomeric proteins or DSB repair proteins affects the frequency of large deletions.
In Aim 1 C we will characterize the DSB repair proteins involved in the formation of chromosome aberrations using cell clones that contain a GFP gene activated by intrachromosomal rearrangements, and a DsRed gene activated by interchromosomal rearrangemetns.
In Aim 2 A we will use a novel real-time quantitative PCR assay for chromosome healing to monitor how knockdown of telomeric proteins or ATM affects the frequency of chromosome healing in isogenic cell clones generated by moving a telomere to a location adjacent to the I-SceI site using Cre/LoxP-mediated recombination.
In Aim 2 B we will compare the appearance of PIF1 helicase, a protein known to inhibit chromosome healing in yeast, at subtelomeric and interstitial DSBs with and without knockdown of TRF2 or ATM, using the same cell clones containing the DSBs marked with the LacI-GFP fusion protein used in Aim 1A.

Public Health Relevance

Chromosome instability is an important factor in promoting the multiple genetic changes leading to cancer. We have demonstrated that one mechanism for chromosome instability in human cancer cells involves the sudden loss of telomeres, the caps that protect the ends of chromosomes and prevent chromosome fusion. This proposal will investigate the mechanisms for two factors that we have shown are important in this process. The first is the sensitivity of regions near telomeres to double-strand breaks, which we believe is an important factor in the increased rate of telomere loss in cancer cells. The second is restoration of lost telomeres, called chromosome healing, which we have shown can prevent chromosome instability due to telomere loss. Understanding the mechanisms of telomere loss and chromosome healing in human cancer cells may lead to new approaches for the limiting the chromosome instability responsible for cancer cell progression and resistance to anti-cancer therapies.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01CA120205-09
Application #
8676447
Study Section
Radiation Therapeutics and Biology Study Section (RTB)
Program Officer
Pelroy, Richard
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California San Francisco
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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Muraki, Keiko; Han, Limei; Miller, Douglas et al. (2013) The role of ATM in the deficiency in nonhomologous end-joining near telomeres in a human cancer cell line. PLoS Genet 9:e1003386
Murnane, John P (2012) Telomere dysfunction and chromosome instability. Mutat Res 730:28-36
Miller, Douglas; Reynolds, Gloria E; Mejia, Ricardo et al. (2011) Subtelomeric regions in mammalian cells are deficient in DNA double-strand break repair. DNA Repair (Amst) 10:536-44
Kulkarni, Avanti; Zschenker, Oliver; Reynolds, Gloria et al. (2010) Effect of telomere proximity on telomere position effect, chromosome healing, and sensitivity to DNA double-strand breaks in a human tumor cell line. Mol Cell Biol 30:578-89
Murnane, John P (2010) Telomere loss as a mechanism for chromosome instability in human cancer. Cancer Res 70:4255-9
Zschenker, Oliver; Kulkarni, Avanti; Miller, Douglas et al. (2009) Increased sensitivity of subtelomeric regions to DNA double-strand breaks in a human cancer cell line. DNA Repair (Amst) 8:886-900