Fission Yeast Tel1ATM and Rad3ATR Promote Telomere Protection and Telomerase Recruitment

PLoS Genetics, Aug 2009

The checkpoint kinases ATM and ATR are redundantly required for maintenance of stable telomeres in diverse organisms, including budding and fission yeasts, Arabidopsis, Drosophila, and mammals. However, the molecular basis for telomere instability in cells lacking ATM and ATR has not yet been elucidated fully in organisms that utilize both the telomere protection complex shelterin and telomerase to maintain telomeres, such as fission yeast and humans. Here, we demonstrate by quantitative chromatin immunoprecipitation (ChIP) assays that simultaneous loss of Tel1ATM and Rad3ATR kinases leads to a defect in recruitment of telomerase to telomeres, reduced binding of the shelterin complex subunits Ccq1 and Tpz1, and increased binding of RPA and homologous recombination repair factors to telomeres. Moreover, we show that interaction between Tpz1-Ccq1 and telomerase, thought to be important for telomerase recruitment to telomeres, is disrupted in tel1Δ rad3Δ cells. Thus, Tel1ATM and Rad3ATR are redundantly required for both protection of telomeres against recombination and promotion of telomerase recruitment. Based on our current findings, we propose the existence of a regulatory loop between Tel1ATM/Rad3ATR kinases and Tpz1-Ccq1 to ensure proper protection and maintenance of telomeres in fission yeast.

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Fission Yeast Tel1ATM and Rad3ATR Promote Telomere Protection and Telomerase Recruitment

Nakamura TM (2009) Fission Yeast Tel1ATM and Rad3ATR Promote Telomere Protection and Telomerase Recruitment. PLoS Genet 5(8): e1000622. doi:10.1371/journal.pgen.1000622 ATM ATR Fission Yeast Tel1 and Rad3 Promote Telomere Protection and Telomerase Recruitment Bettina A. Moser 0 Lakxmi Subramanian 0 Lyne Khair 0 Ya-Ting Chang 0 Toru M. Nakamura 0 Nancy Maizels, University of Washington, United States of America 0 Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago , Chicago, Illinois , United States of America The checkpoint kinases ATM and ATR are redundantly required for maintenance of stable telomeres in diverse organisms, including budding and fission yeasts, Arabidopsis, Drosophila, and mammals. However, the molecular basis for telomere instability in cells lacking ATM and ATR has not yet been elucidated fully in organisms that utilize both the telomere protection complex shelterin and telomerase to maintain telomeres, such as fission yeast and humans. Here, we demonstrate by quantitative chromatin immunoprecipitation (ChIP) assays that simultaneous loss of Tel1ATM and Rad3ATR kinases leads to a defect in recruitment of telomerase to telomeres, reduced binding of the shelterin complex subunits Ccq1 and Tpz1, and increased binding of RPA and homologous recombination repair factors to telomeres. Moreover, we show that interaction between Tpz1-Ccq1 and telomerase, thought to be important for telomerase recruitment to telomeres, is disrupted in tel1D rad3D cells. Thus, Tel1ATM and Rad3ATR are redundantly required for both protection of telomeres against recombination and promotion of telomerase recruitment. Based on our current findings, we propose the existence of a regulatory loop between Tel1ATM/Rad3ATR kinases and Tpz1-Ccq1 to ensure proper protection and maintenance of telomeres in fission yeast. - Funding: This work was supported by UIC start-up funds, the Sidney Kimmel Scholar Program, and NIH grant GM078253 to TMN. LS is supported by a predoctoral fellowship from the American Heart Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Telomeres, the nucleoprotein protective structures at ends of eukaryotic chromosomes, are essential for stable maintenance of eukaryotic genomes [1]. In most eukaryotic species, telomeric DNA is made up of short repetitive G-rich sequences that can be extended by the specialized reverse transcriptase telomerase, to overcome the inability of semi-conservative DNA replication machineries to fully replicate ends of linear DNA [2]. While most of the telomeric G-rich repeats are composed of double-stranded DNA (dsDNA), telomeres end with G-rich 39 single-stranded DNA (ssDNA), known as G-tail. Both dsDNA and ssDNA portions are important for maintaining functional telomeres as they provide binding sites for telomeric repeat sequence-specific binding proteins, as well as various DNA repair and checkpoint proteins, that are critical for proper maintenance of telomeres. In mammalian cells, the shelterin complex, composed of TRF1, TRF2, TIN2, RAP1, TPP1 and POT1, plays critical roles in the stable maintenance of telomeres [1]. TRF1 and TRF2 bind specifically to telomeric dsDNA G-rich repeats via their C-terminal myb-like DNA binding domain, while POT1 binds to the telomeric G-tail via its N-terminal OB-fold domains [1]. On the other hand, RAP1, despite the fact that it is evolutionarily related to the budding yeast dsDNA telomeric repeat-binding protein Rap1, cannot directly bind to DNA, and it is recruited to telomeres via its interaction with TRF2 [1]. Likewise, TIN2 is recruited to telomeres by its ability to interact with both TRF1 and TRF2 [3]. TIN2 plays a central role in the formation of the shelterin complex through its ability to interact with the POT1 binding partner TPP1. Previous studies have shown that TRF2 is essential for preventing fusion of telomeres by non-homologous end-joining (NHEJ) and for attenuating ATM-dependent checkpoint signaling [4]. On the other hand, POT1 is critical for protection of telomeres against nucleolytic processing and for attenuating ATR-dependent checkpoint signaling [4]. The POT1-TPP1 sub-complex was also found to interact with the telomerase complex and to increase processivity of telomerase [5,6]. Fission yeast Schizosaccharomyces pombe is an attractive model system for understanding how the shelterin complex contributes to telomere function since this organism utilizes proteins that show a high degree of conservation to the mammalian shelterin subunits [7]. In contrast, the more extensively studied budding yeast Saccharomyces cerevisiae, while providing unparalleled detailed molecular understanding on how telomere maintenance is regulated, cannot provide much insight into how the shelterin components might contribute to telomere (...truncated)


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Bettina A. Moser, Lakxmi Subramanian, Lyne Khair, Ya-Ting Chang, Toru M. Nakamura. Fission Yeast Tel1ATM and Rad3ATR Promote Telomere Protection and Telomerase Recruitment, PLoS Genetics, 2009, 8, DOI: 10.1371/journal.pgen.1000622