Telomere stability plays an important role in the preservation of genomic stability and is maintained through the coordinated actions of telomere-specific proteins and DNA repair and replication proteins [1, 2]. Flap endonuclease 1 (FEN1) is a protein that plays a role in lagging-strand DNA replication, base excision repair, homologous recombination, and reinitiation of stalled replication forks [3, 4]. Here, we demonstrate that FEN1 depletion leads to telomere dysfunction characterized by the presence of gammaH2AX and sister telomere loss. Expression of catalytically active telomerase, the reverse transcriptase that adds telomeric repeats to chromosome ends, was sufficient to rescue telomere dysfunction upon FEN1 depletion. Strikingly, FEN1 depletion exclusively abrogates telomeres replicated by lagging-strand DNA replication. Genetic rescue experiments utilizing FEN1 mutant proteins that retained the ability to localize to telomeric repeats revealed that FEN1's nuclease activity and ability to interact with the Werner protein (WRN) and telomere-binding protein (TRF2) were required for FEN1 activity at the telomere. Given FEN1's role in lagging-strand DNA replication and reinitiation of stalled replication forks, we propose that FEN1 contributes to telomere stability by ensuring efficient telomere replication.

Telomeres are terminal repetitive DNA sequences whose stability requires the coordinated actions of telomere-binding proteins and the DNA replication and repair machinery. Recently, we demonstrated that the DNA replication and repair protein Flap endonuclease 1 (FEN1) is required for replication of lagging strand telomeres. Here, we demonstrate for the first time that FEN1 is required for efficient re-initiation of stalled replication forks. At the telomere, we find that FEN1 depletion results in replicative stress as evidenced by fragile telomere expression and sister telomere loss. We show that FEN1 participation in Okazaki fragment processing is not required for efficient telomere replication. Instead we find that FEN1 gap endonuclease activity, which processes DNA structures resembling stalled replication forks, and the FEN1 interaction with the RecQ helicases are vital for telomere stability. Finally, we find that FEN1 depletion neither impacts cell cycle progression nor in vitro DNA replication through non-telomeric sequences. Our finding that FEN1 is required for efficient replication fork re-initiation strongly suggests that the fragile telomere expression and sister telomere losses observed upon FEN1 depletion are the direct result of replication fork collapse. Together, these findings suggest that other nucleases compensate for FEN1 loss throughout the genome during DNA replication but fail to do so at the telomere. We propose that FEN1 maintains stable telomeres by facilitating replication through the G-rich lagging strand telomere, thereby ensuring high fidelity telomere replication.

Background:Telomere fragility occurs following replication stress. Results: Leading strand-specific telomere fragility is induced by FEN1 depletion and transcription inhibition and is rescued by ectopic RNase H1 expression. Conclusion: RNA:DNA hybrids contribute to telomere fragility that is limited by FEN1. Significance: This is the first explanation for leading strand-specific telomere fragility and is the first leading strand-specific role for FEN1.

Abrogation of telomere stability through loss-of-function mutations in telomere binding proteins contributes to genomic instability and cancer progression. Recently, Flap endonuclease 1 (FEN1) was shown to contribute to telomere stability in human cells that had not yet activated a telomere maintenance mechanism, suggesting that abrogation of FEN1 function influences the transformation process by compromising telomere stability and driving genomic instability. Here, we analyse the telomeres in human cancer cells following FEN1 depletion. We show that FEN1 is required for telomere stability in cells that rely on the alternative lengthening of telomere (ALT) mechanism. Indeed, FEN1 depletion resulted in telomere dysfunction, characterized by formation of telomere dysfunction-induced foci (TIFs) and end-to-end fusions in ALT-positive cells. In contrast, no telomere phenotype was observed in telomerase-positive cells on FEN1 depletion, suggesting that ongoing telomerase activity protected telomeres. In consonance with this, we found that expression of the catalytic component of telomerase (hTERT) but not an inactive allele rescued telomere dysfunction on FEN1 depletion in ALT cells. Our data suggest that mutations that arise in FEN1 affect telomere stability and genome fidelity by promoting telomere fusions and anaphase-bridge-breakage cycles, which further drive genome instability and thereby contribute to the transformation process.

Chirality is a very important characteristic of optically active molecules and polyaromatics with helical structures, and plays a vital role in various applications in material science. In the present work, we show the effects of fluorine substitution at various positions in a figure-8-shaped [5]helicene dimer on the ground and excited state g-factors. Calculations for the ground and excited states are performed at the MP2 and ADC(2) levels of theory, respectively. The results reveal that fluorination has a large effect on the excited state structures. The values of the excited state dissymmetry factors for the molecules with fluorinations at both ends of the figure-8 systems are smaller than that of the parent system. On the other hand, fluorinations only in the stacked-phenyl region results in an increase in the value of g cpl � � � � . The perfluorinated system shows the smallest

Ligand-induced receptor dimerization is the first functional step in receptor signaling, representing the most proximal, functional read-out for receptor activation. Here we present a novel application of the Enzyme Fragment Complementation system to monitor receptor-receptor interactions at the surface of intact cells, applicable to diverse receptor types such as RTKs, Interleukin receptors, BMP receptors and cytokine receptors, amongst others. For the purpose of this poster, we will focus on the HER family of receptors. It is well understood that the HER family proteins can dimerize with the other members of its family leading to a complicated oncogenic signaling cascade. Surprisingly, existing cellular assays have been unable to faithfully monitor these interactions proximally in a drug discovery setting. We present EFC-based cellular assays that monitor EGFR homodimerization, EGFR-ErbB2 and ErbB2-ErbB3 heterodimerization events at the receptor. The high signal to noise ratio, serum tolerance and reproducibility make these assays ideal for a diverse range of applications for the identification and development of therapeutic small molecules and biologics, including screening, functional characterization, QC lot release assays and neutralizing antibody studies. Citation Format: Jane Lamerdin, Abhishek Saharia, Jennifer Lin-Jones, Mimi Nguyen, Hyna Fabionar, Sangeetha Gunturi, Abha Srivastava, Tom Wehrman. Detection of EGFR, HER2, HER3 and HER4 homodimerization and heterodimerization using EFC-based cellular assays. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3742. doi:10.1158/1538-7445.AM2014-3742

No abstract