Rif1 maintains telomeres and mediates DNA repair by encasing DNA ends

In yeast, Rif1 is part of the telosome, where it inhibits telomerase and checkpoint signaling at chromosome ends. In mammalian cells, Rif1 is not telomeric, but it suppresses DNA end resection at chromosomal breaks, promoting repair by nonhomologous end joining (NHEJ). Here, we describe crystal structures for the uncharacterized and conserved ∼125-kDa N-terminal domain of Rif1 from Saccharomyces cerevisiae (Rif1-NTD), revealing an α-helical fold shaped like a shepherd's crook. We identify a high-affinity DNA-binding site in the Rif1-NTD that fully encases DNA as a head-to-tail dimer. Engagement of the Rif1-NTD with telomeres proved essential for checkpoint control and telomere length regulation. Unexpectedly, Rif1-NTD also promoted NHEJ at DNA breaks in yeast, revealing a conserved role of Rif1 in DNA repair. We propose that tight associations between the Rif1-NTD and DNA gate access of processing factors to DNA ends, enabling Rif1 to mediate diverse telomere maintenance and DNA repair functions

Human Papillomavirus-16 E7 Interacts with Glutathione S-Transferase P1 and Enhances Its Role in Cell Survival

Background:Human Papillomavirus (HPV)-16 is a paradigm for "high-risk" HPVs, the causative agents of virtually all cervical carcinomas. HPV E6 and E7 viral genes are usually expressed in these tumors, suggesting key roles for their gene products, the E6 and E7 oncoproteins, in inducing malignant transformation.Methodology/Principal Findings:By protein-protein interaction analysis, using mass spectrometry, we identified glutathione S-transferase P1-1 (GSTP1) as a novel cellular partner of the HPV-16 E7 oncoprotein. Following mapping of the region in the HPV-16 E7 sequence that is involved in the interaction, we generated a three-dimensional molecular model of the complex between HPV-16 E7 and GSTP1, and used this to engineer a mutant molecule of HPV-16 E7 with strongly reduced affinity for GSTP1.When expressed in HaCaT human keratinocytes, HPV-16 E7 modified the equilibrium between the oxidized and reduced forms of GSTP1, thereby inhibiting JNK phosphorylation and its ability to induce apoptosis. Using GSTP1-deficient MCF-7 cancer cells and siRNA interference targeting GSTP1 in HaCaT keratinocytes expressing either wild-type or mutant HPV-16 E7, we uncovered a pivotal role for GSTP1 in the pro-survival program elicited by its binding with HPV-16 E7.Conclusions/Significance:This study provides further evidence of the transforming abilities of this oncoprotein, setting the groundwork for devising unique molecular tools that can both interfere with the interaction between HPV-16 E7 and GSTP1 and minimize the survival of HPV-16 E7-expressing cancer cells. © 2009 Mileo et al

Nuclear architecture organized by Rif1 underpins the replication-timing program

DNA replication is temporally and spatially organized in all eukaryotes, yet the molecular control and biological function of the replication-timing program are unclear. Rif1 is required for normal genome-wide regulation of replication timing, but its molecular function is poorly understood. Here we show that in mouse embryonic stem cells, Rif1 coats late-replicating domains and, with Lamin B1, identifies most of the late-replicating genome. Rif1 is an essential determinant of replication timing of non-Lamin B1-bound late domains. We further demonstrate that Rif1 defines and restricts the interactions between replication-timing domains during the G1 phase, thereby revealing a function of Rif1 as organizer of nuclear architecture. Rif1 loss affects both number and replication-timing specificity of the interactions between replication-timing domains. In addition, during the S phase, Rif1 ensures that replication of interacting domains is temporally coordinated. In summary, our study identifies Rif1 as the molecular link between nuclear architecture and replication-timing establishment in mammals

Rif1 Binding and Control of Chromosome-Internal DNA Replication Origins Is Limited by Telomere Sequestration

The Saccharomyces cerevisiae telomere-binding protein Rif1 plays an evolutionarily conserved role in control of DNA replication timing by promoting PP1-dependent dephosphorylation of replication initiation factors. However, ScRif1 binding outside of telomeres has never been detected, and it has thus been unclear whether Rif1 acts directly on the replication origins that it controls. Here, we show that, in unperturbed yeast cells, Rif1 primarily regulates late-replicating origins within 100 kb of a telomere. Using the chromatin endogenous cleavage ChEC-seq technique, we robustly detect Rif1 at late-replicating origins that we show are targets of its inhibitory action. Interestingly, abrogation of Rif1 telomere association by mutation of its Rap1-binding module increases Rif1 binding and origin inhibition elsewhere in the genome. Our results indicate that Rif1 inhibits replication initiation by interacting directly with origins and suggest that Rap1-dependent sequestration of Rif1 increases its effective concentration near telomeres, while limiting its action at chromosome-internal sites

The effect of the shortest telomere on cell proliferation

International audienceProgress in understanding telomere replication and its relationship to senescence has been hampered by the intrinsic variations in telomeres and the stochastic nature of senescence onset. The work in our lab relies on our ability to circumvent and experimentally dissect the causes of this heterogeneity. Our strategy is to manipulate and track single telomeres in individual cells and experimentally evaluate the effects on proliferation potential of single cell lines to build a mathematical model of senescence.We therefore set up a microfluidics-based live cell imaging assay to study replicative senescence in single Saccharomyces cerevisiae cell lineages after telomerase inactivation. We found that most lineages undergo an abrupt and irreversible transition consistent with a mathematical model in which the first telomere reaching a short critical length triggers the onset of senescence. Other cells exhibit transient checkpoint-dependent cell cycle delays followed by normal cell cycles before senescence. We have now shown that these two pathways to senescence correspond to two kinetically and mechanistically distinct, age-dependent processes underlying non- terminal and terminal senescence arrest.Taking advantage of our novel FinalCut system, we also investigated the proliferation potential of telomerase-negative cells with critically short telomeres of different defined lengths. Our results are consistent with a model whereby a small portion of cells may enter senescence in a probabilistic manner as the length of the shortest telomere decreases. Our results thus directly demonstrate that the length of the shortest telomere is a major determinant of entry into senescence, but a hidden parameter may also contribute to the onset of senescence upon telomerase inactivation. However, when the shortest telomere reaches a critical length, all cells immediately arrest. In addition, we found that at this critical length, the shortest telomere can be stably maintained without signs of fusions or degradations. This suggests that telomeres can be both critically short and functional with respect to telomere protection

The effect of the shortest telomere on cell proliferation

International audienceProgress in understanding telomere replication and its relationship to senescence has been hampered by the intrinsic variations in telomeres and the stochastic nature of senescence onset. The work in our lab relies on our ability to circumvent and experimentally dissect the causes of this heterogeneity. Our strategy is to manipulate and track single telomeres in individual cells and experimentally evaluate the effects on proliferation potential of single cell lines to build a mathematical model of senescence.We therefore set up a microfluidics-based live cell imaging assay to study replicative senescence in single Saccharomyces cerevisiae cell lineages after telomerase inactivation. We found that most lineages undergo an abrupt and irreversible transition consistent with a mathematical model in which the first telomere reaching a short critical length triggers the onset of senescence. Other cells exhibit transient checkpoint-dependent cell cycle delays followed by normal cell cycles before senescence. We have now shown that these two pathways to senescence correspond to two kinetically and mechanistically distinct, age-dependent processes underlying non- terminal and terminal senescence arrest.Taking advantage of our novel FinalCut system, we also investigated the proliferation potential of telomerase-negative cells with critically short telomeres of different defined lengths. Our results are consistent with a model whereby a small portion of cells may enter senescence in a probabilistic manner as the length of the shortest telomere decreases. Our results thus directly demonstrate that the length of the shortest telomere is a major determinant of entry into senescence, but a hidden parameter may also contribute to the onset of senescence upon telomerase inactivation. However, when the shortest telomere reaches a critical length, all cells immediately arrest. In addition, we found that at this critical length, the shortest telomere can be stably maintained without signs of fusions or degradations. This suggests that telomeres can be both critically short and functional with respect to telomere protection

How the shortest telomere in the cell signals senescence

International audienceTelomeres ensure genome integrity and are maintained by telomerase. During replicative senescence, telomerase is inactivated, telomere sequences progressively shorten and set the limit for cell proliferation. When telomeres shorten, they are thought to lose their protective caps at a critical short length, which activates the DNA damage response and recruits DNA damage repair activities that would degrade, fuse, or recombine dysfunctional telomeres. However, the structure(s) of short and dysfunctional telomeres, which respectively trigger permanent replicative senescence or potentially promote genome instability, remain unclear.To define the structure of telomeres at the point of dysfunction and the fate of cells carrying them, we have developed in Saccharomyces cerevisiae a system called FinalCut to induce a single telomere of defined length in cells in which we can conditionally inactivate telomerase. This allows structural analysis of this telomere and, combined with the use of our microfluidic system to track consecutive cell cycles from telomerase inactivation to cell death, we can achieve single telomere and single cell resolution. Our results show that cells reach senescence earlier and more synchronously, as the shortest telomere is set at a shorter length. This is in agreement with the first telomere reaching a short length being sufficient to trigger senescence in budding yeast. Below a certain threshold, the shorter telomere becomes unstable and degrades. However, we can define a length for which a short telomere appears to be stable, and the cells stop dividing at the same time. Combined with a mathematical model of senescence, our results suggest that the probability of a telomere signaling senescence increases as it shortens, but at low frequencies. When a telomere reaches a critical length, it can be stably maintained in cells, while activating the DNA damage checkpoint, without causing obvious fusions or degradation. The structure of this critically short telomere will be discussed