Biology subjects

Morris, D. K.

Publications and source records attributed to Morris, D. K..

3 recordsLinked to original sources

bioRxiv2023

Hyperextended telomeres promote C-circle formation in telomerase positive human cells

Telomere length maintenance is crucial to cancer cell immortality. Up to 15% of cancers utilize a telomerase-independent, recombination-based mechanism termed alternative lengthening of telomeres (ALT). The primary ALT biomarker is the C-circle, a type of circular DNA with extrachromosomal telomere repeats (cECTRs). How C-circles form is not well characterized. To investigate C-circle formation in telomerase+ cells, we studied the human cen3tel cell line, in which telomeres progressively hyper-elongated post TERT-immortalization. cECTR signal was observed in 2D gels and C-circle assays but not t-circle assays, which also detect cECTRs. Telomerase activity and C-circle signal were not separable in the analysis of clonal populations, consistent with C-circle production occurring within telomerase+ cells. Two other long telomere, telomerase+ (LTT+) cell lines, HeLa1.3 (~23 kb telomeres) and HeLaE1 (~50 kb telomeres), had similar cECTR properties. Telomerase activity did not directly impact C-circle signal in LTT+ cells; instead, C-circle signal correlated with telomere length. LTT+ lines were less sensitive to hydroxyurea than an ALT+ cell line, suggesting that ALT status is a stronger contributor to replication stress levels than telomere length. Additionally, FANCM did not suppress C-circles in LTT+ cells as it does in ALT+ cells. Thus, C-circle formation may be driven by telomere length, independently of telomerase and replication stress, highlighting limitations of C-circles as a stand-alone ALT biomarker.

cancer biology↗

bioRxiv2019

The DNA end-binding protein Ku associates with human telomeres primarily via protein-protein interactions

The Ku heterodimer (Ku70/Ku80) binds DNA ends with high affinity but without sequence specificity and, upon binding ends created by double-stranded breaks (DSBs), initiates canonical nonhomologous end-joining (c-NHEJ). Ku also localizes to functional telomeres where its c-NHEJ activity is inhibited. Interestingly, Ku has been co-opted at telomeres across species, where it performs varied telomeric functions. In humans, Ku is essential for its role in telomere maintenance, but how it associates with human telomeres is not known. Analysis of Kus telomere association in different populations of cen3tel cells, which had a wide range of average telomere lengths, supported Kus localization at human telomeres primarily via protein-protein interaction. We also found that the Ku70 and Ku80 5 helices, which are on opposing sides of the heterodimer and were previously implicated in Saccharomyces cerevisiae Kus NHEJ and telomeric functions, respectively, participated in Kus telomere association in human cells. While the Ku70 5 mutant showed increased interaction with TRF2, the Ku80 5 mutant was not impacted for TRF2 association. Interestingly, residues altered to impair Kus DNA end-binding function were also involved in TRF2 interaction and telomere association. Overall, our results suggest protein-protein interactions as the primary mode by which Ku associates with human telomeres.

molecular biology↗

bioRxiv2019

Loss of Ku’s DNA end binding activity affects telomere length via destabilizing telomere-bound Est1 rather than altering TLC1 homeostasis

Saccharomyces cerevisiae telomerase, which maintains telomere length, is comprised of an RNA component, TLC1, the reverse transcriptase, Est2, and regulatory subunits, including Est1. The Yku70/Yku80 (Ku) heterodimer, a DNA end binding (DEB) protein, also contributes to telomere length maintenance. Ku binds TLC1 and telomere ends in a mutually exclusive fashion, and is required to maintain levels and nuclear localization of TLC1. Ku also interacts with Sir4, which localizes to telomeres. Here we sought to determine the role of Kus DEB activity in telomere length maintenance by utilizing yku70-R456E mutant strains, in which Ku has reduced DEB and telomere association but proficiency in TLC1 and Sir4 binding, and TLC1 nuclear retention. Telomere lengths in a yku70-R456E strain were nearly as short as those in yku{Delta} strains and shorter than in strains lacking either Sir4, Ku:Sir4 interaction, or Ku:TLC1 interaction. TLC1 levels were decreased in the yku70-R456E mutant, yet overexpression of TLC1 failed to restore telomere length. Reduced DEB activity did not impact Est1s ability to associate with telomerase but did result in decreased association of Est1 with the telomere. These findings suggest Kus DEB activity maintains telomere length homeostasis by preserving Est1s interaction at the telomere rather than altering TLC1 levels.

molecular biology↗