Carolyn Mary Price

CST (CTC1-STN1-TEN1) is a telomere associated complex that binds ssDNA and is required for multiple steps in telomere replication, including termination of G-strand extension by telomerase and synthesis of the complementary C-strand. CST contains seven OB-folds which appear to mediate CST function by modulating CST binding to ssDNA and the ability of CST to recruit or engage partner proteins. However, the mechanism whereby CST achieves its various functions remains unclear. To address the mechanism, we generated a series of CTC1 mutants and studied their effect on CST binding to ssDNA and their ability to rescue CST function in CTC1^−/− cells. We identified the OB-B domain as a key determinant of telomerase termination but not C-strand synthesis. CTC1-ΔB expression rescued C-strand fill-in, prevented telomeric DNA damage signaling and growth arrest. However, it caused progressive telomere elongation and the accumulation of telomerase at telomeres, indicating an inability to limit telomerase action. The CTC1-ΔB mutation greatly reduced CST-TPP1 interaction but only modestly affected ssDNA binding. OB-B point mutations also weakened TPP1 association, with the deficiency in TPP1 interaction tracking with an inability to limit telomerase action. Overall, our results indicate that CTC1-TPP1 interaction plays a key role in telomerase termination.

Human CST (CTC1-STN1-TEN1) is an RPA-like complex that associates with G-rich single-strand DNA and helps resolve replication problems both at telomeres and genome-wide. We previously showed that CST binds and disrupts G-quadruplex (G4) DNA in vitro, suggesting that CST may prevent in vivo blocks to replication by resolving G4 structures. Here, we demonstrate that CST binds and unfolds G4 with similar efficiency to RPA. In cells, CST is recruited to telomeric and non-telomeric chromatin upon G4 stabilization, even when ATR/ATM pathways were inhibited. STN1 depletion increases G4 accumulation and slows bulk genomic DNA replication. At telomeres, combined STN1 depletion and G4 stabilization causes multi-telomere FISH signals and telomere loss, hallmarks of deficient telomere duplex replication. Strand-specific telomere FISH indicates preferential loss of C-strand DNA while analysis of BrdU uptake during leading and lagging-strand telomere replication shows preferential under-replication of lagging telomeres. Together these results indicate a block to Okazaki fragment synthesis. Overall, our findings indicate a novel role for CST in maintaining genome integrity through resolution of G4 structures both ahead of the replication fork and on the lagging strand template.

Telomerase elongates the telomeric G-strand to prevent telomere shortening through conventional DNA replication. However, synthesis of the complementary C-strand by DNA polymerase α is also required to maintain telomere length. Polymerase α cannot perform this role without the ssDNA binding complex CST (CTC1-STN1-TEN1). Here we describe the roles of individual CST subunits in telomerase regulation and G-overhang maturation in human colon cancer cells. We show that CTC1-STN1 limits telomerase action to prevent G-overhang overextension. CTC1^−/− cells exhibit telomeric DNA damage and growth arrest due to overhang elongation whereas TEN1^−/− cells do not. However, TEN1 is essential for C-strand synthesis and TEN1^−/− cells exhibit progressive telomere shortening. DNA binding analysis indicates that CTC1-STN1 retains affinity for ssDNA but TEN1 stabilizes binding. We propose CTC1-STN1 binding is sufficient to terminate telomerase action but altered DNA binding dynamics renders CTC1-STN1 unable to properly engage polymerase α on the overhang for C-strand synthesis.

The sex chromosomes are enriched with germline genes that are activated during the late stages of spermatogenesis. Due to meiotic sex chromosome inactivation (MSCI), these sex chromosome-linked genes must escape silencing for activation in spermatids, thereby ensuring their functions for male reproduction. RNF8, a DNA damage response protein, and SCML2, a germline-specific Polycomb protein, are two major, known regulators of this process. Here, we show that RNF8 and SCML2 cooperate to regulate ubiquitination during meiosis, an early step to establish active histone modifications for subsequent gene activation. Double mutants of Rnf8 and Scml2 revealed that RNF8-dependent monoubiquitination of histone H2A at Lysine 119 (H2AK119ub) is deubiquitinated by SCML2, demonstrating interplay between RNF8 and SCML2 in ubiquitin regulation. Additionally, we identify distinct functions of RNF8 and SCML2 in the regulation of ubiquitination: SCML2 deubiquitinates RNF8-independent H2AK119ub but does not deubiquitinate RNF8-dependent polyubiquitination. RNF8-dependent polyubiquitination is required for the establishment of H3K27 acetylation, a marker of active enhancers, while persistent H2AK119ub inhibits establishment of H3K27 acetylation. Following the deposition of H3K27 acetylation, H3K4 dimethylation is established as an active mark on poised promoters. Together, we propose a model whereby regulation of ubiquitin leads to the organization of poised enhancers and promoters during meiosis, which induce subsequent gene activation from the otherwise silent sex chromosomes in postmeiotic spermatids. To produce unimpaired sperm, precise activation of germline-specific genes is an essential step during the late stages of spermatogenesis. However, sex chromosomes carrying these genes become silenced in a chromosome-wide manner during meiosis in a process called meiotic sex chromosome inactivation. Sex chromosome inactivation is maintained from meiosis into postmeiotic spermatids. Thus, to ensure the function of sex chromosome-linked (sex-linked) genes required for male reproduction, these genes must escape silencing for activation in spermatids. Here, we unravel the epigenetic mechanisms that underlie the activation of sex-linked genes from otherwise inactive sex chromosomes in the male germline. We determine the mechanism by which two factors regulate gene activation: one is RNF8, a DNA damage response protein, and the other is SCML2, a germline-specific Polycomb protein. Our data suggest that, during meiosis, RNF8 and SCML2 cooperate to regulate ubiquitination, which establishes active epigenetic modifications on enhancers and promoters for subsequent gene activation; these memories are maintained through meiotic divisions to induce gene activation in spermatids. Importantly, this study uncovers novel epigenetic mechanisms that underlie specific gene activation in spermatids and illuminates potential causes of male infertility.

Human CST (CTC1–STN1–TEN1) is a ssDNA-binding complex that helps resolve replication problems both at telomeres and genome-wide. CST resembles Replication Protein A (RPA) in that the two complexes harbor comparable arrays of OB-folds and have structurally similar small subunits. However, the overall architecture and functions of CST and RPA are distinct. Currently, the mechanism underlying CST action at diverse replication issues remains unclear. To clarify CST mechanism, we examined the capacity of CST to bind and resolve DNA structures found at sites of CST activity. We show that CST binds preferentially to ss-dsDNA junctions, an activity that can explain the incremental nature of telomeric C-strand synthesis following telomerase action. We also show that CST unfolds G-quadruplex structures, thus providing a mechanism for CST to facilitate replication through telomeres and other GC-rich regions. Finally, smFRET analysis indicates that CST binding to ssDNA is dynamic with CST complexes undergoing concentration-dependent self-displacement. These findings support an RPA-based model where dissociation and re-association of individual OB-folds allow CST to mediate loading and unloading of partner proteins to facilitate various aspects of telomere replication and genome-wide resolution of replication stress.

To prevent progressive telomere shortening as a result of conventional DNA replication, new telomeric DNA must be added onto the chromosome end. The de novo DNA synthesis involves elongation of the G-rich strand of the telomere by telomerase. In human cells, the CST complex (CTC1-STN1-TEN1) also functions in telomere replication. CST first aids in duplication of the telomeric dsDNA. Then after telomerase has extended the G-rich strand, CST facilitates fill-in synthesis of the complementary C-strand. Here, we analyze telomere structure after disruption of human CTC1 and demonstrate that functional CST is essential for telomere length maintenance due to its role in mediating C-strand fill-in. Removal of CTC1 results in elongation of the 3΄ overhang on the G-rich strand. This leads to accumulation of RPA and telomeric DNA damage signaling. G-overhang length increases with time after CTC1 disruption and at early times net G-strand growth is apparent, indicating telomerase-mediated G-strand extension. In contrast, C-strand length decreases continuously, indicating a deficiency in C-strand fill-in synthesis. The lack of C-strand maintenance leads to gradual shortening of the telomeric dsDNA, similar to that observed in cells lacking telomerase. Thus, telomerase-mediated G-strand extension and CST-mediated C-strand fill-in are equally important for telomere length maintenance.

Mammalian CST (CTC1-STN1-TEN1) participates in multiple aspects of telomere replication and genome-wide recovery from replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Conservation between CTC1 and RPA1 is less apparent. Currently the mechanism underlying CST action is largely unknown. Here we address CST mechanism by using a DNA-binding mutant, (STN1 OB-fold mutant, STN1-OBM) to examine the relationship between DNA binding and CST function. In vivo, STN1-OBM affects resolution of endogenous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. These selective effects indicate mechanistic differences in CST action during resolution of different replication problems. In vitro binding studies show that STN1 directly engages both short and long ssDNA oligonucleotides, however STN1-OBM preferentially destabilizes binding to short substrates. The finding that STN1-OBM affects binding to only certain substrates starts to explain the in vivo separation of function observed in STN1-OBM expressing cells. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve different replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The in vitro studies also provide insight into CST binding mechanism. Like RPA, CST likely contacts DNA via multiple OB folds. However, the importance of STN1 for binding short substrates indicates differences in the architecture of CST and RPA DNA-protein complexes. Based on our results, we propose a dynamic DNA binding model that provides a general mechanism for CST action at diverse forms of replication stress. Mammalian CST (CTC1/STN1/TEN1) is a three protein complex that aids in several steps during telomere replication and has genome-wide roles during recovery from replication fork stalling. Loss of CST leads to abnormalities in telomere structure, genomic instability and defects in chromosome segregation. Currently, we do not understand how CST acts to ensure the resolution of very diverse types of replication problem. We set out to address this question by studying a mutant form of CST that was predicted to alter DNA binding. The mutations are in the STN1 subunit. In vivo, the STN1 mutant (STN1-OBM) affects some aspects of CST function while others are normal. The effects of STN1-OBM do not align with the telomeric versus non-telomeric roles of CST but instead separate out different aspects of CST function at telomeres and genome-wide. In vitro binding studies indicate that STN1-OBM disrupts binding to only short DNA substrates. Since CST is likely to encounter DNA substrates of varied length and structure in vivo as it helps resolve different replication problems, this finding starts to explain why STN1-OBM affects only certain aspects of CST function. Our in vitro binding studies also shed light on how CST actually binds to DNA and they suggest a novel “dynamic binding model” that provides a mechanistic explanation for how CST helps resolve a diverse array of replication problems to preserve genome stability.

Tetrahymena telomeres are protected by a protein complex composed of Pot1, Tpt1, Pat1, and Pat2. Pot1 binds the 3′ overhang and serves multiple roles in telomere maintenance. Here we describe Pot2, a paralog of Pot1 which has evolved a novel function during Tetrahymena sexual reproduction. Pot2 is unnecessary for telomere maintenance during vegetative growth, as the telomere structure is unaffected by POT2 macronuclear gene disruption. Pot2 is expressed only in mated cells, where it accumulates in developing macronuclei around the time of two chromosome processing events: internal eliminated sequence (IES) excision and chromosome breakage. Chromatin immunoprecipitation (ChIP) demonstrated Pot2 localization to regions of chromosome breakage but not to telomeres or IESs. Pot2 association with chromosome breakage sites (CBSs) occurs slightly before chromosome breakage. Pot2 did not bind CBSs or telomeric DNA in vitro, suggesting that it is recruited to CBSs by another factor. The telomere proteins Pot1, Pat1, and Tpt1 and the IES binding factor Pdd1 fail to colocalize with Pot2. Thus, Pot2 is the first protein found to associate specifically with CBSs. The selective association of Pot2 versus Pdd1 with CBSs or IESs indicates a mechanistic difference between the chromosome processing events at these two sites. Moreover, ChIP revealed that histone marks characteristic of IES processing, H3K9me3 and H3K27me3, are absent from CBSs. Thus, the mechanisms of chromosome breakage and IES excision must be fundamentally different. Our results lead to a model where Pot2 directs chromosome breakage by recruiting telomerase and/or the endonuclease responsible for DNA cleavage to CBSs.

Mammalian CST (CTC1-STN1-TEN1) is a telomere-associated complex that functions in telomere duplex replication and fill-in synthesis of the telomeric C-strand following telomerase action. CST also facilitates genome-wide replication recovery after HU-induced fork stalling by increasing origin firing. CTC1 and STN1 were originally isolated as a DNA polymerase α stimulatory factor. Here we explore how CST abundance affects recovery from drugs that cause different types of DNA damage and replication stress. We show that recovery from HU and aphidicolin induced replication stress is increased by CST over-expression. Elevated CST increases dNTP incorporation and origin firing after HU release and decreases the incidence of anaphase bridges and micronuclei after aphidicolin removal. While the frequency of origin firing after HU release is proportional to CST abundance, the number of cells entering S-phase to initiate replication is unchanged by CST overexpression or STN1 depletion. Instead the CST-relate...

Although studies with the ciliate Tetrahymena thermophila have played a central role in advancing our understanding of telomere biology and telomerase mechanisms and composition, the full complement of Tetrahymena telomere proteins has not yet been identified. Previously, we demonstrated that in Tetrahymena, the telomeric 3′ overhang is protected by a three-protein complex composed of Pot1a, Tpt1, and Pat1. Here we show that Tpt1 and Pat1 associate with a fourth protein, Pat2 (Pot1 associated Tetrahymena 2). Mass spectrometry of proteins copurifying with Pat1 or Tpt1 identified peptides from Pat2, Pot1a, Tpt1, and Pat1. The lack of other proteins copurifying with Pat1 or Tpt1 implies that the overhang is protected by a four-protein Pot1a-Tpt1-Pat1-Pat2 complex. We verified that Pat2 localizes to telomeres, but we were unable to detect direct binding to telomeric DNA. Cells depleted of Pat2 continue to divide, but the telomeres exhibit gradual shortening. The lack of growth arrest indicates that, in contrast to Pot1a and Tpt1, Pat2 is not required for the sequestration of the telomere from the DNA repair machinery. Instead, Pat2 is needed to regulate telomere length, most likely by acting in conjunction with Pat1 to allow telomerase access to the telomere.