Deregulated DNA ADP-ribosylation impairs telomere replication.

The recognition that DNA can be ADP ribosylated provides an unexpected regulatory level of how ADP-ribosylation contributes to genome stability, epigenetics and immunity. Yet, it remains unknown whether DNA ADP-ribosylation (DNA-ADPr) promotes genome stability and how it is regulated. Here, we show that telomeres are subject to DNA-ADPr catalyzed by PARP1 and removed by TARG1. Mechanistically, we show that DNA-ADPr is coupled to lagging telomere DNA strand synthesis, forming at single-stranded DNA present at unligated Okazaki fragments and on the 3' single-stranded telomere overhang. Persistent DNA-linked ADPr, due to TARG1 deficiency, eventually leads to telomere shortening. Furthermore, using the bacterial DNA ADP-ribosyl-transferase toxin to modify DNA at telomeres directly, we demonstrate that unhydrolyzed DNA-linked ADP-ribose compromises telomere replication and telomere integrity. Thus, by identifying telomeres as chromosomal targets of PARP1 and TARG1-regulated DNA-ADPr, whose deregulation compromises telomere replication and integrity, our study highlights and establishes the critical importance of controlling DNA-ADPr turnover for sustained genome stability.

Extrachromosomal telomere DNA derived from excessive strand displacements.

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication, evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), including C-circles, are unique to ALT cells, their generation process remains undefined. Here, we introduce a method to detect single-stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single-stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear and circular C-rich ssDNAs are generated concurrently. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.

Extrachromosomal Telomeres Derived from Excessive Strand Displacements.

Alternative Lengthening of Telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication (BIR), evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), unique to ALT cells, are considered potential precursors of C-circles, their generation process remains undefined. Here, we introduce a highly sensitive method to detect single stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear C-rich ssDNA accumulation may indeed precede C-circle formation. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a new model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.

Mechanisms of insertions at a DNA double-strand break.

Insertions and deletions (indels) are common sources of structural variation, and insertions originating from spontaneous DNA lesions are frequent in cancer. We developed a highly sensitive assay called insertion and deletion sequencing (Indel-seq) to monitor rearrangements in human cells at the TRIM37 acceptor locus that reports indels stemming from experimentally induced and spontaneous genome instability. Templated insertions, which derive from sequences genome wide, require contact between donor and acceptor loci, require homologous recombination, and are stimulated by DNA end-processing. Insertions are facilitated by transcription and involve a DNA/RNA hybrid intermediate. Indel-seq reveals that insertions are generated via multiple pathways. The broken acceptor site anneals with a resected DNA break or invades the displaced strand of a transcription bubble or R-loop, followed by DNA synthesis, displacement, and then ligation by non-homologous end joining. Our studies identify transcription-coupled insertions as a critical source of spontaneous genome instability that is distinct from cut-and-paste events.

The Molecular Mechanisms and Therapeutic Prospects of Alternative Lengthening of Telomeres (ALT).

As detailed by the end replication problem, the linear ends of a cell's chromosomes, known as telomeres, shorten with each successive round of replication until a cell enters into a state of growth arrest referred to as senescence. To maintain their immortal proliferation capacity, cancer cells must employ a telomere maintenance mechanism, such as telomerase activation or the Alternative Lengthening of Telomeres pathway (ALT). With only 10-15% of cancers utilizing the ALT mechanism, progress towards understanding its molecular components and associated hallmarks has only recently been made. This review analyzes the advances towards understanding the ALT pathway by: (1) detailing the mechanisms associated with engaging the ALT pathway as well as (2) identifying potential therapeutic targets of ALT that may lead to novel cancer therapeutic treatments. Collectively, these studies indicate that the ALT molecular mechanisms involve at least two distinct pathways induced by replication stress and damage at telomeres. We suggest exploiting tumor dependency on ALT is a promising field of study because it suggests new approaches to ALT-specific therapies for cancers with poorer prognosis. While substantial progress has been made in the ALT research field, additional progress will be required to realize these advances into clinical practices to treat ALT cancers and improve patient prognoses.

Proliferation of adult human bronchial epithelial cells without a telomere maintenance mechanism for over 200 population doublings.

To date, there is no direct evidence of telomerase activity in adult lung epithelial cells, but typical culture conditions only support cell proliferation for 30-40 population doublings (PD), a point at which telomeres remain relatively long. Here we report that in in vitro low stress culture conditions consisting of a fibroblast feeder layer, rho-associated coiled coil protein kinase inhibitor (ROCKi), and low oxygen (2%), normal human bronchial epithelial basal progenitor cells (HBECs) divide for over 200 PD without engaging a telomere maintenance mechanism (almost four times the "Hayflick limit"). HBECs exhibit critically short telomeres at 200 PD and the population of cells start to undergo replicative senescence. Subcloning these late passage cells to clonal density, to mimic lung injury in vivo, selects for rare subsets of HBECs that activate low levels of telomerase activity to maintain short telomeres. CRISPR/Cas9 knockout of human telomerase reverse transcriptase or treatment with the telomerase-mediated telomere targeting agent 6-thio-2'deoxyguanosine abrogates colony growth in these late passage cultures (>200 PD) but not in early passage cultures (<200 PD). To our knowledge, this is the first study to report such long-term growth of HBECs without a telomere maintenance mechanism. This report also provides direct evidence of telomerase activation in HBECs near senescence when telomeres are critically short. This novel cell culture system provides an experimental model to understand how telomerase is regulated in normal adult tissues.

Clustered telomeres in phase-separated nuclear condensates engage mitotic DNA synthesis through BLM and RAD52.

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. One of the hallmarks of ALT cancer is the excessively clustered telomeres in promyelocytic leukemia (PML) bodies, represented as large bright telomere foci. Here, we present a model system that generates telomere clustering in nuclear polySUMO (small ubiquitin-like modification)/polySIM (SUMO-interacting motif) condensates, analogous to PML bodies, and thus artificially engineered ALT-associated PML body (APB)-like condensates in vivo. We observed that the ALT-like phenotypes (i.e., a small fraction of heterogeneous telomere lengths and formation of C circles) are rapidly induced by introducing the APB-like condensates together with BLM through its helicase domain, accompanied by ssDNA generation and RPA accumulation at telomeres. Moreover, these events lead to mitotic DNA synthesis (MiDAS) at telomeres mediated by RAD52 through its highly conserved N-terminal domain. We propose that the clustering of large amounts of telomeres in human cancers promotes ALT that is mediated by MiDAS, analogous to Saccharomyces cerevisiae type II ALT survivors.

Brca2 abrogation engages with the alternative lengthening of telomeres via break-induced replication.

A subset of cancer cells maintains their telomeres without telomerase through the recombination-based alternative lengthening of telomeres (ALT) pathway. Currently, it is not yet clear in what context ALT is induced and how the pathway choice is made. Here, we show that abrogation of Brca2 reinforces break-induced replication (BIR) and engages with ALT pathway. Brca2 depletion in telomerase-null mouse cells alleviated the growth defect, accompanied by telomere elongation, suggesting the induction of ALT. We also found that Brca2-depleted telomerase-null cells exhibited dynamic clustering of telomeres from G2 phase in Promyelocytic Nuclear (PML) bodies. For Brca2-deficient ALT induction, Rad51 was dispensable but Mre11 and Rad52 were required. Congruently, conservative telomeric DNA synthesis was apparent in mitosis, indicating that the absence of Brca2 directed towards Rad52-mediated BIR. Collectively, we propose that Brca2 abrogation can instigate ALT tumourigenesis through the induction of BIR. This study implies that inhibitors of BIR may be useful for BRCA2-associated ALT-type cancers. Assessing ALT features may be considered for the tailored therapy of BRCA2-associated cancers.

Truncated Adenomatous Polyposis Coli Mutation Induces Asef-Activated Golgi Fragmentation.

Adenomatous polyposis coli (APC) is a key molecule to maintain cellular homeostasis in colonic epithelium by regulating cell-cell adhesion, cell polarity, and cell migration through activating the APC-stimulated guanine nucleotide-exchange factor (Asef). The APC-activated Asef stimulates the small GTPase, which leads to decreased cell-cell adherence and cell polarity, and enhanced cell migration. In colorectal cancers, while truncated APC constitutively activates Asef and promotes cancer initiation and progression, regulation of Asef by full-length APC is still unclear. Here, we report the autoinhibition mechanism of full-length APC. We found that the armadillo repeats in full-length APC interact with the APC residues 1362 to 1540 (APC-2,3 repeats), and this interaction competes off and inhibits Asef. Deletion of APC-2,3 repeats permits Asef interactions leading to downstream signaling events, including the induction of Golgi fragmentation through the activation of the Asef-ROCK-MLC2. Truncated APC also disrupts protein trafficking and cholesterol homeostasis by inhibition of SREBP2 activity in a Golgi fragmentation-dependent manner. Our study thus uncovers the autoinhibition mechanism of full-length APC and a novel gain of function of truncated APC in regulating Golgi structure, as well as cholesterol homeostasis, which provides a potential target for pharmaceutical intervention against colon cancers.

Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes.

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Saccharomyces cerevisiae Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer.

Alternative lengthening of telomeres can be maintained by preferential elongation of lagging strands.

Alternative lengthening of telomeres (ALT) is a telomerase independent telomere maintenance mechanism that occurs in ∼15% of cancers. The potential mechanism of ALT is homology-directed telomere synthesis, but molecular mechanisms of how ALT maintains telomere length in human cancer is poorly understood. Here, we generated TERC (telomerase RNA) gene knockouts in telomerase positive cell lines that resulted in long-term surviving clones acquiring the ALT pathway but at a very low frequency. By comparing these ALT cells with parental telomerase positive cells, we observed that ALT cells possess excessively long telomeric overhangs derived from telomere elongation processes that mostly occur during S phase. ALT cells exhibited preferential elongation of the telomeric lagging strands, whereas telomerase positive cells exhibited similar elongation between leading and lagging strands. We propose that the ALT pathway preferentially occurs at telomeric lagging strands leading to heterogeneous telomere lengths observed in most ALT cancers.

Regulation of the Human Telomerase Gene TERT by Telomere Position Effect-Over Long Distances (TPE-OLD): Implications for Aging and Cancer.

Telomerase is expressed in early human development and then becomes silenced in most normal tissues. Because ~90% of primary human tumors express telomerase and generally maintain very short telomeres, telomerase is carefully regulated, particularly in large, long-lived mammals. In the current report, we provide substantial evidence for a new regulatory control mechanism of the rate limiting catalytic protein component of telomerase (hTERT) that is determined by the length of telomeres. We document that normal, young human cells with long telomeres have a repressed hTERT epigenetic status (chromatin and DNA methylation), but the epigenetic status is altered when telomeres become short. The change in epigenetic status correlates with altered expression of TERT and genes near to TERT, indicating a change in chromatin. Furthermore, we identified a chromosome 5p telomere loop to a region near TERT in human cells with long telomeres that is disengaged with increased cell divisions as telomeres progressively shorten. Finally, we provide support for a role of the TRF2 protein, and possibly TERRA, in the telomere looping maintenance mechanism through interactions with interstitial TTAGGG repeats. This provides new insights into how the changes in genome structure during replicative aging result in an increased susceptibility to age-related diseases and cancer prior to the initiation of a DNA damage signal.

TERT Promoter Mutations Enhance Telomerase Activation by Long-Range Chromatin Interactions.

Although single-nucleotide somatic mutations in the proximal promoter of the human telomerase reverse transcriptase (TERT) gene create novel consensus sequences for transcription factors that enhance TERT expression, the precise mechanism of how telomerase is reactivated in cancer cells remains poorly understood. In this issue, Akincilar and colleagues identify a potential mechanism of TERT reactivation that is mediated by a novel long-range chromatin interaction between the TERT promoter on chromosome 5p and a 300-kb upstream region. This permits recruitment of the transcription factor GABPA in mutant TERT promoters but not in wild-type promoters. Cancer Discov; 6(11); 1212-4. ©2016 AACRSee related article by Akincilar and colleagues, p. 1276.

Paradoxical delay of senescence upon depletion of BRCA2 in telomerase-deficient worms.

BRCA2 is a multifunctional tumor suppressor involved in homologous recombination (HR), mitotic checkpoint regulation, and telomere homeostasis. Absence of Brca2 in mice results in progressive shortening of telomeres and senescence, yet cells are prone to neoplastic transformation with elongated telomeres, suggesting that BRCA2 has positive and negative effects on telomere length regulation along the path to tumorigenesis. Using Caenorhabditis elegans as a model, we show here that depletion of BRC-2, an ortholog of BRCA2, paradoxically delays senescence in telomerase-deficient mutant worms. Telomerase-deficient worms (trt-1) exhibit early replication senescence due to short telomeres. It should be noted that worms mutated in brc-2 are not viable as well due to massive genotoxic insults. However, when BRC-2 is depleted by RNA interference in trt-1 mutant worms, the number of generations is unexpectedly increased with telomere length maintained, compared to telomerase mutants. Interestingly, depletion of other HR genes such as rad-51 and rad-54 exhibited similar effects. In worms doubly deficient of telomerase and brc-2, rad-51, or rad-54, extra telomeric C-circles were generated, suggesting that abrogation of HR induces an alteration in telomere environment favorable to illegitimate telomere maintenance when telomerase is absent. Collectively, absence of BRC-2 in telomerase-deficient background first leads to telomere shortening, followed by an induction of an as-yet-unknown telomere maintenance pathway, resulting in delay of senescence. The results have implications in the understanding of dysfunctional BRCA2-associated tumorigenesis.

Loss of BubR1 acetylation causes defects in spindle assembly checkpoint signaling and promotes tumor formation.

BubR1 acetylation is essential in mitosis. Mice heterozygous for the acetylation-deficient BubR1 allele (K243R/+) spontaneously developed tumors with massive chromosome missegregations. K243R/+ mouse embryonic fibroblasts (MEFs) exhibited a weakened spindle assembly checkpoint (SAC) with shortened mitotic timing. The generation of the SAC signal was intact, as Mad2 localization to the unattached kinetochore (KT) was unaltered; however, because of the premature degradation of K243R-BubR1, the mitotic checkpoint complex disassociated prematurely in the nocodazole-treated condition, suggesting that maintenance of the SAC is compromised. BubR1 acetylation was also required to counteract excessive Aurora B activity at the KT for stable chromosome-spindle attachments. The association of acetylation-deficient BubR1 with PP2A-B56α phosphatase was reduced, and the phosphorylated Ndc80 at the KT was elevated in K243R/+ MEFs. In relation, there was a marked increase of micronuclei and p53 mutation was frequently detected in primary tumors of K243R/+ mice. Collectively, the combined effects of failure in chromosome-spindle attachment and weakened SAC cause genetic instability and cancer in K243R/+ mice.

The breast cancer susceptibility gene BRCA2 is required for the maintenance of telomere homeostasis.

Inactivating mutations in the breast cancer susceptibility gene BRCA2 cause gross chromosomal rearrangements. Chromosome structural instability in the absence of BRCA2 is thought to result from defective homology-directed DNA repair. Here, we show that BRCA2 links the fidelity of telomere maintenance with genetic integrity. Absence of BRCA2 resulted in signs of dysfunctional telomeres, such as telomere shortening, erosions, and end fusions in proliferating mouse fibroblasts. BRCA2 localized to the telomeres in S phase in an ATR-dependent manner, and its absence resulted in the accumulation of common fragile sites, particularly at the G-rich lagging strand, and increased the telomere sister chromatid exchange in unchallenged cells. The incidence of common fragile sites and telomere sister chromatid exchange increased markedly after treatment with replication inhibitors. Congruently, telomere-induced foci were frequently observed in the absence of Brca2, denoting activation of the DNA damage response and abnormal chromosome end joining. These telomere end fusions constituted a significant portion of chromosome aberrations in Brca2-deficient cells. Our results suggest that BRCA2 is required for telomere homeostasis and may be particularly important for the replication of G-rich telomeric lagging strands.

Identification of Rad51 regulation by BRCA2 using Caenorhabditis elegans BRCA2 and bimolecular fluorescence complementation analysis.

BRCA2 is involved in double-stranded DNA break repair by binding and regulating Rad51-mediated homologous recombination. Insights as to how BRCA2 regulates Rad51-mediated DNA repair arose from in vitro biochemical studies on fragments of BRCA2. However, the large 400-kDa BRCA2 protein has hampered our ability to understand the entire process by which full-length BRCA2 regulates Rad51. Here, we show that CeBRC-2, which is only one tenth the size of mammalian BRCA2, complemented BRCA2-deficiency in Rad51 regulation. CeBRC-2 was able to bind to mammalian Rad51 (mRad51) and form distinct nuclear foci when they interacted. In our bimolecular fluorescence complementation analysis (BiFC), we show that the strength of the interaction between CeBRC-2 and mRad51 increased markedly after DNA damage. The BRC motif of CeBRC-2 was responsible for binding mRad51, but without the OB fold, the complex was unable to target damaged DNA. When CeBRC-2 was introduced into BRCA2-deficient cells, it restored Rad51 foci after DNA damage. Our study suggests a mode of action for BRCA2 with regard to DNA repair.