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The phosphatidylinositol 3′ kinase (PI3K)‐related kinase ATR is crucial for mammalian meiosis. ATR promotes meiotic progression by coordinating key events in DNA repair, meiotic sex chromosome inactivation (MSCI), and checkpoint-dependent quality control during meiotic prophase I. Despite its central roles in meiosis, the ATR-dependent meiotic signaling network remains largely unknown. Here, we used phosphoproteomics to define ATR signaling events in testes from mice following chemical and genetic ablation of ATR signaling. Quantitative analysis of phosphoproteomes obtained after germ cell-specific genetic ablation of the ATR activating 9-1-1 complex or treatment with ATR inhibitor identified over 14,000 phosphorylation sites from testes samples, of which 401 phosphorylation sites were found to be dependent on both the 9-1-1 complex and ATR. Our analyses identified ATR-dependent phosphorylation events in crucial DNA damage signaling and DNA repair proteins including TOPBP1, SMC3, MDC1, RAD50, and SLX4. Importantly, we identified ATR and RAD1-dependent phosphorylation events in proteins involved in mRNA regulatory processes, including SETX and RANBP3, whose localization to the sex body was lost upon ATR inhibition. In addition to identifying the expected ATR-targeted S/T-Q motif, we identified enrichment of an S/T-P-X-K motif in the set of ATR-dependent events, suggesting that ATR promotes signaling via proline-directed kinase(s) during meiosis. Indeed, we found that ATR signaling is important for the proper localization of CDK2 in spermatocytes. Overall, our analysis establishes a map of ATR signaling in mouse testes and highlights potential meiotic-specific actions of ATR during prophase I progression.

Simple Summary Testicular germ cell tumors (TGCTs) are the most common solid malignancy in young men. Fortunately, these tumors are highly curable with conventional genotoxic chemotherapy. However, because these chemotherapeutics have significant short- and long-term side effects, less toxic treatment options are desired. We report that thioridazine, an FDA-approved antipsychotic, differentiates embryonal carcinoma (EC) cells, the cancer stem cells of many malignant TGCTs, and suppresses their tumor propagating activity. Additionally, thioridazine treatment of mice with EC-containing neoplasms extends survival relative to untreated control mice. These findings identify thioridazine as a promising alternative or adjuvant to chemotherapy for the treatment of TGCTs. Abstract Testicular germ cell tumors (TGCTs) are exceptionally sensitive to genotoxic chemotherapy, resulting in a high cure rate for the young men presenting with these malignancies. However, this treatment is associated with significant toxicity, and a subset of malignant TGCTs demonstrate chemoresistance. Mixed nonseminomas often contain pluripotent embryonal carcinoma (EC) cells, the cancer stem cells (CSCs) of these tumors. We hypothesized that differentiation therapy, a treatment strategy which aims to induce differentiation of tumor-propagating CSCs to slow tumor growth, could effectively treat mixed nonseminomas without significant toxicity. The FDA-approved antipsychotic thioridazine and the agricultural antibiotic salinomycin are two drugs previously found to selectively target CSCs, and here we report that these agents differentiate EC cells in vitro and greatly reduce their tumorigenic potential in vivo. Using a novel transformed induced pluripotent stem cell allograft model and a human xenograft model, we show that thioridazine extends the survival of tumor-bearing mice and can reduce the number of pluripotent EC cells within tumors. These results suggest that thioridazine could be repurposed as an alternative TGCT treatment that avoids the toxicity of conventional chemotherapeutics.

P53 has been implicated in the pathogenesis of obesity and diabetes; however, the mechanisms and tissue sites of action are incompletely defined. Therefore, we investigated the role of hepatocyte p53 in metabolic homeostasis using a hepatocyte-specific p53 knockout mouse model. To gain further mechanistic insight, we studied mice under two complementary conditions of restricted weight gain: vertical sleeve gastrectomy (VSG) or food restriction. VSG or sham surgery was performed in high-fat diet-fed male hepatocyte-specific p53 wild-type and knockout littermates. Sham-operated mice were fed ad libitum or food restricted to match their body weight to VSG-operated mice. Hepatocyte-specific p53 ablation in sham-operated ad libitum-fed mice impaired glucose homeostasis, increased body weight, and decreased energy expenditure without changing food intake. The metabolic deficits induced by hepatocyte-specific p53 ablation were corrected, in part by food restriction, and completely by VSG. Unlike food restriction, VSG corrected the effect of hepatocyte p53 ablation to lower energy expenditure, resulting in a greater improvement in glucose homeostasis compared with food restricted mice. These data reveal an important new role for hepatocyte p53 in the regulation of energy expenditure and body weight and suggest that VSG can improve alterations in energetics associated with p53 dysregulation.

Summary: Testicular germ cell tumors (TGCTs) are among the most responsive solid cancers to conventional chemotherapy. To elucidate the underlying mechanisms, we developed a mouse TGCT model featuring germ cell-specific Kras activation and Pten inactivation. The resulting mice developed malignant, metastatic TGCTs composed of teratoma and embryonal carcinoma, the latter of which exhibited stem cell characteristics, including expression of the pluripotency factor OCT4. Consistent with epidemiological data linking human testicular cancer risk to in utero exposures, embryonic germ cells were susceptible to malignant transformation, whereas adult germ cells underwent apoptosis in response to the same oncogenic events. Treatment of tumor-bearing mice with genotoxic chemotherapy not only prolonged survival and reduced tumor size but also selectively eliminated the OCT4-positive cancer stem cells. We conclude that the chemosensitivity of TGCTs derives from the sensitivity of their cancer stem cells to DNA-damaging chemotherapy. : Using a mouse testicular germ cell tumor model, Pierpont et al. establish that male germ cells are susceptible to malignant transformation during a restricted window of embryonic development. The cancer stem cells of the resulting testicular cancers demonstrate genotoxin hypersensitivity, rendering these malignancies highly responsive to conventional chemotherapy. Keywords: testicular germ cell tumor, TGCT, cancer stem cells, CSCs, chemotherapy, embryonal carcinoma, EC, DNA damage response, DDR

The scaffold protein TOPBP1Dpb11 has been implicated in homologous recombination DNA repair, but its function and mechanism of action remain unclear. Liu et al. define a conserved role for TOPBP1Dpb11 in recombination control through regulated, opposing interactions with pro- and anti-resection factors., Genome maintenance and cancer suppression require homologous recombination (HR) DNA repair. In yeast and mammals, the scaffold protein TOPBP1Dpb11 has been implicated in HR, although its precise function and mechanism of action remain elusive. In this study, we show that yeast Dpb11 plays an antagonistic role in recombination control through regulated protein interactions. Dpb11 mediates opposing roles in DNA end resection by coordinating both the stabilization and exclusion of Rad9 from DNA lesions. The Mec1 kinase promotes the pro-resection function of Dpb11 by mediating its interaction with the Slx4 scaffold. Human TOPBP1Dpb11 engages in interactions with the anti-resection factor 53BP1 and the pro-resection factor BRCA1, suggesting that TOPBP1 also mediates opposing functions in HR control. Hyperstabilization of the 53BP1–TOPBP1 interaction enhances the recruitment of 53BP1 to nuclear foci in the S phase, resulting in impaired HR and the accumulation of chromosomal aberrations. Our results support a model in which TOPBP1Dpb11 plays a conserved role in mediating a phosphoregulated circuitry for the control of recombinational DNA repair.

The professional training of cancer researchers in the basic sciences rarely involves interactions with patients. To provide nascent cancer scientists with an appreciation for and experience in interacting with the people most vested in their work, we created a program at Cornell University in which cancer researchers in training engage with the local patient community. Through this program, trainees gain a broader understanding of cancer, beyond the fundamental biology, and learn to effectively communicate scientific information to the public. We find that trainees and community members both benefit from interacting with one another and learning together about cancer using a common language.

Eukaryotic cells continuously experience DNA damage that can perturb key molecular processes like DNA replication. DNA replication forks that encounter DNA lesions typically slow and may stall, which can lead to highly detrimental fork collapse if appropriate protective measures are not executed. Stabilization and protection of stalled replication forks ensures the possibility of effective fork restart and prevents genomic instability. Recent efforts from multiple laboratories have highlighted several proteins involved in replication fork remodeling and DNA damage response pathways as key regulators of fork stability. Homologous recombination factors such as RAD51, BRCA1, and BRCA2, along with components of the Fanconi Anemia pathway, are now known to be crucial for stabilizing stalled replication forks and preventing nascent strand degradation. Several checkpoint proteins have additionally been implicated in fork protection. Ongoing work in this area continues to shed light on a sophisticated molecular pathway that balances the action of DNA resection and fork protection to maintain genomic integrity, with important implications for the fate of both normal and malignant cells following replication stress.

Since the origins of DNA-based life, the enzyme ribonucleotide reductase (RNR) has spurred proliferation because of its rate-limiting role in de novo deoxynucleoside-triphosphate (dNTP) biosynthesis. Paradoxically, the large subunit, RNR-α, of this obligatory two-component complex in mammals plays a context-specific anti-proliferative role. There is little explanation for this dichotomy. Here, we show that RNR-αhas a previously-unrecognized DNA-replication-inhibition function, leading to growth retardation. This underappreciated biological activity functions in the nucleus where RNR-α interacts with ZRANB3. This process suppresses ZRANB3’s function in unstressed cells that we show to be promotion of DNA-synthesis. This non-reductase-function of RNR-α is promoted by RNR-α-hexamerization—induced by natural- and synthetic-nucleotide of dA/ClF/CLA/FLU—which elicits rapid RNR-α nuclear import. The newly-discovered nuclear signaling axis is a primary defense against elevated/imbalanced dNTP-pools that can exert mutagenic effects irrespective of the cell cycle.

Sirt5, localized in the mitochondria, is a member of sirtuin family of NAD(+)-dependent deacetylases. Sirt5 was shown to deacetylate and activate carbamoyl phosphate synthase 1. Most recently, Sirt5 was reported to be the predominant protein desuccinylase and demalonylase in the mitochondria because the ablation of Sirt5 enhanced the global succinylation and malonylation of mitochondrial proteins, including many metabolic enzymes. In order to determine the physiological role of Sirt5 in metabolic homeostasis, we generated a germline Sirt5 deficient (Sirt5(-/-)) mouse model and performed a thorough metabolic characterization of this mouse line. Although a global protein hypersuccinylation and elevated serum ammonia during fasting were observed in our Sirt5(-/-) mouse model, Sirt5 deficiency did not lead to any overt metabolic abnormalities under either chow or high fat diet conditions. These observations suggest that Sirt5 is likely to be dispensable for the metabolic homeostasis under the basal conditions.

The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1., Author Summary Meiosis is a specialized cell division process in which germ cells undergo two cell divisions to produce haploid progeny. Two processes, genetic recombination and chromosome pairing/synapsis, are critical for successful meiosis and the production of gametes with high chromosomal integrity. The RAD9-RAD1-HUS1 (9-1-1) complex has been proposed to play critical roles in recombination as well as in the checkpoint-dependent monitoring of chromosomal synapsis by facilitating activation of the ATR checkpoint kinase. Our data indicate that HUS1 is required for normal germ cell development and fertility, for efficient completion of a subset of meiotic DNA recombination events, and for proper exclusion of the non-sex chromosomes from a specialized, repressive chromatin domain containing the X and Y chromosomes. However, HUS1 is not required for the meiotic functions of ATR in responding to chromosome synapsis defects. Furthermore, RAD1 localizes to sites along asynapsed chromosomes that lack detectable RAD9, and does so in the absence of Hus1, implicating RAD1 in a novel response to unsynapsed chromatin that is independent of the canonical 9-1-1 complex. Since mice lacking Hus1 in germ cells exhibit chromosomal abnormalities and severely reduced fertility, this work has broad implications for the maintenance of genome stability in the germline and for human reproductive health.

Invariant NKT (iNKT) cells play important roles in the immune response. ITK and TXK/RLK are Tec family kinases that are expressed in iNKT cells; the expression level of ITK is ∼7-fold higher than that of TXK. Itk−/− mice have reduced iNKT cell frequency and numbers, with defects in development and cytokine secretion that are exacerbated in Itk/Txk double-knockout mice. In contrast, there is no iNKT cell defect in Txk−/− mice. To determine whether ITK and TXK play distinct roles in iNKT cell development and function, we examined mice that overexpress TXK in T cells at levels similar to Itk. Overexpression of TXK rescues the maturation and cytokine secretion of Itk−/− iNKT cells, as well as altered expression of transcription factors T-bet, eomesodermin, and PLZF. In contrast, the increased apoptosis observed in Itk−/− splenic iNKT cells is not affected by TXK overexpression, likely due to the lack of effect on the elevated expression of p53 regulated proapoptotic pathways Fas, Bax, and Bad in those cells. Supporting this idea, p53−/− and Bax−/− mice have increased splenic iNKT cells. Our results suggest that TXK plays an overlapping role with ITK in iNKT cell development and function but that ITK also has a unique function in the survival of iNKT cells, likely via a p53-dependent pathway.

Limitations of current medical procedures for detecting early lung cancers inspire the need for new diagnostic imaging modalities for the direct microscopic visualization of lung nodules. Multiphoton microscopy (MPM) provides for subcellular resolution imaging of intrinsic fluorescence from unprocessed tissue with minimal optical attenuation and photodamage. We demonstrate that MPM detects morphological and spectral features of lung tissue and differentiates between normal, inflammatory and neoplastic lung. Ex vivo MPM imaging of intrinsic two-photon excited fluorescence was performed on mouse and canine neoplastic, inflammatory and tumor-free lung sites. Results showed that MPM detected microanatomical differences between tumor-free and neoplastic lung tissue similar to standard histopathology but without the need for tissue processing. Furthermore, inflammatory sites displayed a distinct red-shifted fluorescence compared to neoplasms in both mouse and canine lung, and adenocarcinomas displayed a less pronounced fluorescence emission in the 500 to 550 nm region compared to adenomas in mouse models of lung cancer. These spectral distinctions were also confirmed by two-photon excited fluorescence microspectroscopy. We demonstrate the feasibility of applying MPM imaging of intrinsic fluorescence for the differentiation of lung neoplasms, inflammatory and tumor-free lung, which motivates the application of multiphoton endoscopy for the in situ imaging of lung nodules.

Cytolethal distending toxin (CDT) is a heterotrimeric AB-type genotoxin produced by several clinically important Gram-negative mucocutaneous bacterial pathogens. Irrespective of the bacterial species of origin, CDT causes characteristic and irreversible cell cycle arrest and apoptosis in a broad range of cultured mammalian cell lineages. The active subunit CdtB has structural homology with the phosphodiesterase family of enzymes including mammalian DNase I, and alone is necessary and sufficient to account for cellular toxicity. Indeed, mammalian cells treated with CDT initiate a DNA damage response similar to that elicited by ionizing radiation-induced DNA double strand breaks resulting in cell cycle arrest and apoptosis. The mechanism of CDT-induced apoptosis remains incompletely understood, but appears to involve both p53-dependent and -independent pathways. While epithelial, endothelial and fibroblast cell lines respond to CDT by undergoing arrest of cell cycle progression resulting in nuclear and cytoplasmic distension that precedes apoptotic cell death, cells of haematopoietic origin display rapid apoptosis following a brief period of cell cycle arrest. In this review, the ecology of pathogens producing CDT, the molecular biology of bacterial CDT and the molecular mechanisms of CDT-induced cytotoxicity are critically appraised. Understanding the contribution of a broadly conserved bacterial genotoxin that blocks progression of the mammalian cell cycle, ultimately causing cell death, should assist with elucidating disease mechanisms for these important pathogens.

The mammalian ortholog of yeast Slx4, BTBD12, is an ATM substrate that functions as a scaffold for various DNA repair activities. Mutations of human BTBD12 have been reported in a new sub-type of Fanconi anemia patients. Recent studies have implicated the fly and worm orthologs, MUS312 and HIM-18, in the regulation of meiotic crossovers arising from double-strand break (DSB) initiating events and also in genome stability prior to meiosis. Using a Btbd12 mutant mouse, we analyzed the role of BTBD12 in mammalian gametogenesis. BTBD12 localizes to pre-meiotic spermatogonia and to meiotic spermatocytes in wildtype males. Btbd12 mutant mice have less than 15% normal spermatozoa and are subfertile. Loss of BTBD12 during embryogenesis results in impaired primordial germ cell proliferation and increased apoptosis, which reduces the spermatogonial pool in the early postnatal testis. During prophase I, DSBs initiate normally in Btbd12 mutant animals. However, DSB repair is delayed or impeded, resulting in persistent γH2AX and RAD51, and the choice of repair pathway may be altered, resulting in elevated MLH1/MLH3 focus numbers at pachynema. The result is an increase in apoptosis through prophase I and beyond. Unlike yeast Slx4, therefore, BTBD12 appears to function in meiotic prophase I, possibly during the recombination events that lead to the production of crossovers. In line with its expected regulation by ATM kinase, BTBD12 protein is reduced in the testis of Atm−/− males, and Btbd12 mutant mice exhibit increased genomic instability in the form of elevated blood cell micronucleus formation similar to that seen in Atm−/− males. Taken together, these data indicate that BTBD12 functions throughout gametogenesis to maintain genome stability, possibly by co-ordinating repair processes and/or by linking DNA repair events to the cell cycle via ATM., Author Summary Mutations in genes essential for genome maintenance during meiosis can result in severe disruptions to spermatogenesis and subsequent low fertility and/or birth defects in mammals. The mammalian homolog of yeast Slx4, BTBD12, plays a critical role in somatic cell repair in mice. Here, we show that this critical function extends to mammalian germ cells, by examining the effects of a Btbd12 gene disruption in mice. Btbd12 mutant mice show severely reduced fertility, as a result of both pre-meiotic spermatogonial proliferation defects and impairment of proper meiotic progression. BTBD12 appears to be required for normal progression of double-strand break repair events that result in the formation of crossovers between maternal and paternal homologous chromosomes, with Btbd12 mutants displaying an increase in unrepaired breaks, impaired homologous chromosome interactions, and a slight increase in the number of crossover intermediates. BTBD12 protein is also down-regulated in the testes of Atm null mice, supporting previous studies showing that BTBD12 is a target of ATM kinase. These data provide new evidence about the role of BTBD12 in mammalian gametogenesis and are critical to furthering the understanding of the molecular processes involved in meiotic DNA repair.

Lung cancer is the leading killer among all cancers for both men and women in the US, and is associated with one of the lowest 5-year survival rates. Current diagnostic techniques, such as histopathological assessment of tissue obtained by computed tomography guided biopsies, have limited accuracy, especially for small lesions. Early diagnosis of lung cancer can be improved by introducing a real-time, optical guidance method based on the in vivo application of multiphoton microscopy (MPM). In particular, we hypothesize that MPM imaging of living lung tissue based on two-photon excited intrinsic fluorescence and second harmonic generation can provide sufficient morphologic and spectroscopic information to distinguish between normal and diseased lung tissue. Here, we used an experimental approach based on MPM with multichannel fluorescence detection for initial discovery that MPM spectral imaging could differentiate between normal and neoplastic lung in ex vivo samples from a murine model of lung cancer. Current results indicate that MPM imaging can directly distinguish normal and neoplastic lung tissues based on their distinct morphologies and fluorescence emission properties in non-processed lung tissue. Moreover, we found initial indication that MPM imaging differentiates between normal alveolar tissue, inflammatory foci, and lung neoplasms. Our long-term goal is to apply results from ex vivo lung specimens to aid in the development of multiphoton endoscopy for in vivo imaging of lung abnormalities in various animal models, and ultimately for the diagnosis of human lung cancer.

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in deoxyribonucleoside triphosphate (dNTP) biosynthesis, with important roles in nuclear genome maintenance. RNR is also essential for maintenance of mitochondrial DNA (mtDNA) in mammals. The mechanisms regulating mtDNA copy number in mammals are only being discovered. In budding yeast, RNR overexpression resulted in increased mtDNA levels, and rescued the disease phenotypes caused by a mutant mtDNA polymerase. This raised the question of whether mtDNA copy number increase by RNR induction could be a strategy for treating diseases with mtDNA mutations. We show here that high-level overexpression of RNR subunits (Rrm1, Rrm2 and p53R2; separately or in different combinations) in mice does not result in mtDNA copy number elevation. Instead, simultaneous expression of two RNR subunits leads to imbalanced dNTP pools and progressive mtDNA depletion in the skeletal muscle, without mtDNA mutagenesis. We also show that endogenous RNR transcripts are downregulated in response to large increases of mtDNA in mice, which is indicative of nuclear-mitochondrial crosstalk with regard to mtDNA copy number. Our results establish that RNR is not limiting for mtDNA copy number in mice, and provide new evidence for the importance of balanced dNTP pools in mtDNA maintenance in postmitotic tissues.

We demonstrate for the first time that a stable, micron-scale segregation of focal enrichments of sterols exists at physiological temperature in the plasma membrane of live murine and human sperm. These enrichments of sterols represent microheterogeneities within this membrane domain overlying the acrosome. Previously, we showed that cholera toxin subunit B (CTB), which binds the glycosphingolipid, GM1, localizes to this same domain in live sperm. Interestingly, the GM1 undergoes an unexplained redistribution upon cell death. We now demonstrate that GM1 is also enriched in the acrosome, an exocytotic vesicle. Transfer of lipids between this and the plasma membrane occurs at cell death, increasing GM1 in the plasma membrane without apparent release of acrosomal contents. This finding provides corroborative support for an emerging model of regulated exocytosis in which membrane communications might occur without triggering the “acrosome reaction.” Comparison of the dynamics of CTB-bound endogenous GM1 and exogenous BODIPY–GM1 in live murine sperm demonstrate that the sub-acrosomal ring (SAR) functions as a specialized diffusion barrier segregating specific lipids within the sperm head plasma membrane. Our data show significant differences between endogenous lipids and exogenous lipid probes in terms of lateral diffusion. Based on these studies, we propose a hierarchical model to explain the segregation of this sterol- and GM1-enriched domain in live sperm, which is positioned to regulate sperm fertilization competence and mediate interactions with the oocyte. Moreover, our data suggest potential origins of subtypes of membrane raft microdomains enriched in sterols and/or GM1 that can be separated biochemically. J. Cell. Physiol. 218: 522–536, 2009. © 2008 Wiley-Liss, Inc.

The Rad9-Rad1-Hus1 (9-1-1) cell cycle checkpoint complex plays a key role in the DNA damage response. Cells with a defective 9-1-1 complex have been shown to be sensitive to apoptosis induced by certain types of genotoxic stress. However, the mechanism linking the loss of a functional 9-1-1 complex to the cell death machinery has yet to be determined. Here, we report that etoposide treatment dramatically upregulates the BH3-only proteins, Bim and Puma, in Hus1-deficient cells. Inhibition of either Bim or Puma expression in Hus1-knockout cells confers significant resistance to etoposide-induced apoptosis, whereas knockdown of both proteins results in further resistance, suggesting that Bim and Puma cooperate in sensitizing Hus1-deficient cells to etoposide treatment. Moreover, we found that Rad9 collaborates with Bim and Puma to sensitize Hus1-deficient cells to etoposide-induced apoptosis. In response to DNA damage, Rad9 localizes to chromatin in Hus1-wild-type cells, whereas in Hus1-deficient cells, it is predominantly located in the cytoplasm where it binds to Bcl-2. Taken together, these results suggest that loss of Hus1 sensitizes cells to etoposide-induced apoptosis not only by inducing Bim and Puma expressions but also by releasing Rad9 into the cytosol to augment mitochondrial apoptosis.

"Natura non facit saltum" (nature makes no leap) the Latins used to say, meaning that nature does not like discontinuities. Cells make no exception and indeed any discontinuity in the DNA double helix is promptly detected, triggering an alteration of cell proliferation and an attempt to repair. Yet, linear chromosomes bear DNA ends that are compatible with normal cell proliferation and they escape, under normal conditions, any repair. How telomeres, the chromosomes tips, achieve that is not fully understood. We recently observed that the Rad9/Hus1/Rad1 (911) complex, previously known for its functions in DNA metabolism and DNA damage responses, is constitutively associated with telomeres and plays an important role in their maintenance. Here, we summarize the available data and discuss the potential mechanisms of 911 action at telomeres.

Mouse Hus1 encodes an evolutionarily conserved DNA damage response protein. In this study we examined how targeted deletion of Hus1 affects cell cycle checkpoint responses to genotoxic stress. Unlike hus1(-) fission yeast (Schizosaccharomyces pombe) cells, which are defective for the G(2)/M DNA damage checkpoint, Hus1-null mouse cells did not inappropriately enter mitosis following genotoxin treatment. However, Hus1-deficient cells displayed a striking S-phase DNA damage checkpoint defect. Whereas wild-type cells transiently repressed DNA replication in response to benzo(a)pyrene dihydrodiol epoxide (BPDE), a genotoxin that causes bulky DNA adducts, Hus1-null cells maintained relatively high levels of DNA synthesis following treatment with this agent. However, when treated with DNA strand break-inducing agents such as ionizing radiation (IR), Hus1-deficient cells showed intact S-phase checkpoint responses. Conversely, checkpoint-mediated inhibition of DNA synthesis in response to BPDE did not require NBS1, a component of the IR-responsive S-phase checkpoint pathway. Taken together, these results demonstrate that Hus1 is required specifically for one of two separable mammalian checkpoint pathways that respond to distinct forms of genome damage during S phase.

SummaryTelomeres, the termini of linear chromosomes, are exceptional in that they are DNA ends that do not normally trigger a DNA-damage response (DDR) and are compatible with normal cellular proliferation. Mammalian telomeres are nevertheless a physiological substrate of the DDR apparatus, as shown by the fact that the inactivation of genes encoding certain DDR factors results in telomere dysfunction [1–3]. However, how DDR factors are integrated with telomere physiology, including telomere length regulation by the specialized reverse transcriptase telomerase, is still largely unclear. Here we report that the mammalian Rad9/Rad1/Hus1 (911) checkpoint complex, which localizes to sites of genome damage and promotes DDR signaling [4], is an integral component of the telomere in human and mouse cells. By the use of quantitative telomere-length measurements, we demonstrate severe telomeric shortening in both Hus1-deficient mouse embryonic fibroblasts and thymocytes from conditional Hus1-knockout mice. We also show that 911 is found in association with catalytically competent telomerase in cell lysates and is a positive regulator of its DNA polymerase activity. These findings identify an unanticipated function for the 911 checkpoint complex at telomeres in mammals and provide a mechanistic link between the activity of DNA-damage-checkpoint proteins and the telomere-maintenance machinery.

The synthesis of dTDP is unique because there is a requirement for thymidylate kinase (TMPK). All other dNDPs including dUDP are directly produced by ribonucleotide reductase (RNR). We report the binding of TMPK and RNR at sites of DNA damage. In tumor cells, when TMPK function is blocked, dUTP is incorporated during DNA double-strand break (DSB) repair. Disrupting RNR recruitment to damage sites or reducing the expression of the R2 subunit of RNR prevents the impairment of DNA repair by TMPK intervention, indicating that RNR contributes to dUTP incorporation during DSB repair. We identified a cell-permeable nontoxic inhibitor of TMPK that sensitizes tumor cells to doxorubicin in vitro and in vivo, suggesting its potential as a therapeutic option.

Skeletal muscle myosin can use a variety of nucleotides to varying effectiveness as substrates for contraction. We previously demonstrated that complete replacement of ATP with 2 deoxy-ATP (dATP) in activation solutions increases contraction of demembranated rabbit skeletal muscle and that enhanced performance results from increased myosin binding and cycling kinetics, especially during sub-maximal calcium activation. Here we report on a transgenic mouse (Tg-RR) that overexpresses the enzyme, ribonucleotide reductase, which converts ADP to dADP (that is rapidly phosphorylated to dATP). This results in a ∼10x increase in dATP content of skeletal muscle, which still constitutes ≤ 1% of the adenosine triphosphate nucleotide pool. We are examining the contractile and metabolic properties of skeletal muscle in this transgenic model. Preliminary data indicates the phosphocreatine to ATP (PCr:ATP) ratio of Tg-RR mice is significantly elevated relative to wild type (Tg-WT) mice, suggesting the Tg-RR mice may have an energetic advantage due to increased high energy phosphate reserves. Furthermore, the Tg-RR mice ran for a longer period of time at increased speed and for longer distances in a graded exercise test. Direct in situ stimulation of the gastrocnemius muscle indicates improved resistance to fatigue, consistent with both the exercise and the metabolic (PCr:ATP) findings. Interestingly, our preliminary experiments suggest the Tg-RR mice produce significantly less peak force and show signs of atrophy. Ongoing experiments are examining changes in mitochondrial content and function as well as contractile properties of isolated muscles to determine the mechanisms underlying the increased fatigue resistance of the Tg-RR mice. These results suggest a new direction for developing interventions to improve exercise tolerance in human patients. Supported by HL11197 (MR).