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PubMed2001

Functional overlap between Sgs1-Top3 and the Mms4-Mus81 endonuclease.

The RecQ DNA helicases, human BLM and yeast Sgs1, form a complex with topoisomerase III (Top3) and are thought to act during DNA replication to restart forks that have paused due to DNA damage or topological stress. We have shown previously that yeast cells lacking SGS1 or TOP3 require MMS4 and MUS81 for viability. Here we show that Mms4 and Mus81 form a heterodimeric structure-specific endonuclease that cleaves branched DNA. Both subunits are required for optimal expression, substrate binding, and nuclease activity. Mms4 and Mus81 are conserved proteins related to the Rad1-Rad10 (XPF/ERCC1) endonuclease required for nucleotide excision repair (NER). However, the Mms4-Mus81 endonuclease is 25 times more active on branched duplex DNA and replication fork substrates than simple Y-forms, the preferred substrate for the NER complexes. We also present genetic data that indicate a novel role for Mms4-Mus81 in meiotic recombination. Our results suggest that stalled replication forks are substrates for Mms4-Mus81 cleavage-particularly in the absence of Sgs1 or BLM. Repair of this double-strand break (DSB) by homologous recombination may be responsible for the elevated levels of sister chromatid exchange (SCE) found in BLM(-/-) cells.

DNA Helicases↗
PubMed2002

Stress-dependent nucleolin mobilization mediated by p53-nucleolin complex formation.

We recently discovered that heat shock causes nucleolin to relocalize from the nucleolus to the nucleoplasm, whereupon it binds replication protein A and inhibits DNA replication initiation. We report that nucleolin mobilization also occurs following exposure to ionizing radiation (IR) and treatment with camptothecin. Mobilization was selective in that another nucleolar marker, upstream binding factor, did not relocalize in response to IR. Nucleolin relocalization was dependent on p53 and stress, the latter initially stimulating nucleolin-p53 complex formation. Nucleolin relocalization and complex formation in vivo were independent of p53 transactivation but required the p53 C-terminal regulatory domain. Nucleolin and p53 also interact directly in vitro, with a similar requirement for p53 domains. These data indicate a novel p53-dependent mechanism in which cell stress mobilizes nucleolin for transient replication inhibition and DNA repair.

Active Transport, Cell Nucleus↗
PubMed2024

RNF4 sustains Myc-driven tumorigenesis by facilitating DNA replication.

The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4-conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.

Animals↗
PubMed2006

Ras induces chromosome instability and abrogation of the DNA damage response.

Ras mutations are frequent in thyroid tumors, the most common endocrine malignancy. The ability of Ras to transform thyroid cells is thought to rely on its mitogenic activity. Unexpectedly, acute expression of activated Ras in normal rat thyroid cells induced a DNA damage response, followed by apoptosis. Notably, a subpopulation of cells evaded apoptosis and emerged with features of transformation, including the loss of epithelial morphology, dedifferentiation, and the acquisition of hormone- and anchorage-independent proliferation. Strikingly, the surviving cells showed marked chromosomal instability. Acutely, Ras stimulated replication stress as evidenced by the induction of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR) activity (Chk1 phosphorylation) and of gammaH2A.X, a marker of DNA damage. Despite the activation of a checkpoint, cells continued through mitosis in the face of DNA damage, resulting in an increase in cells harboring micronuclei, an indication of defects in chromosome segregation and other forms of chromosome damage. Cells that survived exposure to Ras continued to exhibit replication stress (ATR activation) but no longer exhibited gammaH2A.X or full activation of p53. When rechallenged with Ras or DNA-damaging agents, the surviving cells were more resistant to apoptosis than parental cells. These data show that acute expression of activated Ras is sufficient to induce chromosomal instability in the absence of other signals, and suggest that Ras-induced chromosomal instability arises as a consequence of defects in the processing of DNA damage. Hence, abrogation of the DNA damage response may constitute a novel mechanism for Ras transformation.

Animals↗
PubMed2006

Fusion tyrosine kinases: a result and cause of genomic instability.

Reciprocal chromosomal translocations may arise as a result of unfaithful repair of spontaneous DNA double-strand breaks, most probably induced by oxidative stress, radiation, genotoxic chemicals and/or replication stress. Genes encoding tyrosine kinases are targeted by these mechanisms resulting in the generation of chimera genes encoding fusion tyrosine kinases (FTKs). FTKs display transforming activity owing to their constitutive kinase activity causing deregulated proliferation, apoptosis, differentiation and adhesion. Moreover, FTKs are able to facilitate DNA repair, prolong activation of G(2)/M and S cell cycle checkpoints, and elevate expression of antiapoptotic protein Bcl-X(L), making malignant cells less responsive to antitumor treatment. FTKs may also stimulate the generation of reactive oxygen species and enhance spontaneous DNA damage in tumor cells. Unfortunately, FTKs compromise the fidelity of DNA repair mechanisms, which contribute to the accumulation of additional genetic abnormalities leading to the resistance to inhibitors such as imatinib mesylate and malignant progression of the disease.

Animals↗
PubMed2001

[Characterization of the expression and function of SigM an ECF sigma factor in mycobacteria].

The survival of M. tuberculosis within the macrophage depends on its ability to respond to oxidative stress, and the ECF subfamily of sigma factors likely play an important role. We studied SigM, a sigma factor whose gene is located near the origin of DNA replication. In both M. smegmatis and M. bovis BCG, the expression of sigM was induced at high temperature and in stationary phase. Mutants of M. smegmatis without an intact sigM were defective for survival in oxidative stress and also for the induction of thioredoxin reductase activity in oxidative stress. The thioredoxin system reduces disulfide bonds that are formed in oxidative stress. SigM thus appears to regulate thioredoxins and forms part of the bacteria's complex protective responses.

DNA Replication↗
PubMed2005

DNA helicases as targets for anti-cancer drugs.

DNA helicases have essential roles in nucleic acid metabolism by facilitating cellular processes including replication, recombination, DNA repair, and transcription. The vital roles of helicases in these pathways are reflected by their emerging importance in the maintenance of genomic stability. Recently, a number of human diseases with cancer predisposition have been shown to be genetically linked to a specific helicase defect. This has led researchers to further investigate the roles of helicases in cancer biology, and to study the efficacy of targeting human DNA helicases for anti-cancer drug treatment. Helicase-specific inhibition in malignant cells may compromise the high proliferation rates of cancerous tissues. The role of RecQ helicases in response to replicational stress suggests a molecular target for selectively eliminating malignant tumor cells by a cancer chemotherapeutic agent. Alternate DNA secondary structures such as G-quadruplexes that may form in regulatory regions of oncogenes or G-rich telomere sequences are potential targets for cancer therapy since these sequence-specific structures are proposed to affect gene expression and telomerase activation, respectively. Small molecule inhibitors of G-quadruplex helicases may be used to regulate cell cycle progression by modulating promotor activation or disrupting telomere maintenance, important processes of cellular transformation. The design of small molecules which deter helicase function at telomeres may provide a molecular target since telomerase activity is necessary for the proliferation of numerous immortal cells. Although evidence suggests that helicases are specifically inhibited by certain DNA binding compounds, another area of promise in anti-cancer therapy is siRNA technology. Specific knockdown of helicase expression can be utilized as a means to sensitize oncogenic proliferating cell lines. This review will address these topics in detail and summarize the current avenues of research in anti-cancer therapy targeting helicases through small molecule inhibitors of DNA-protein complexes, DNA binding drugs, or down-regulation of helicase gene expression.

Animals↗
PubMed2003

Ectopic hTERT expression extends the life span of human CD4+ helper and regulatory T-cell clones and confers resistance to oxidative stress-induced apoptosis.

Human somatic cells have a limited life span in vitro. Upon aging and with each cell division, shortening of telomeres occurs, which eventually will lead to cell cycle arrest. Ectopic hTERT expression has been shown to extend the life span of human T cells by preventing this telomere erosion. In the present study, we have shown that ectopic hTERT expression extends the life span of CD4+ T helper type 1 or 2 and regulatory T-cell clones and affected neither the in vitro cytokine production profile nor their specificity for antigen. In mixed cell cultures, ectopic hTERT-expressing clones were found to expand in greater numbers than untransduced cells of the same replicative age. This ectopic hTERT-induced growth advantage was not due to an enhanced cell division rate or number of divisions following T-cell receptor-mediated activation, as determined in carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling experiments. Moreover, the susceptibility to activation-induced cell death of both cell types was similar. However, cultures of resting hTERT-transduced T cells contained higher frequencies of Bcl-2-expressing cells and lower active caspase-3-expressing cells, compared with wild-type cells. Furthermore, hTERT-transduced cells were more resistant to oxidative stress, which causes preferential DNA damage in telomeres. Taken together, these results show that ectopic hTERT expression not only protects proliferating T cells from replicative senescence but also confers resistance to apoptosis induced by oxidative stress.

Apoptosis↗
PubMed2005

Inhibition of the herpes simplex virus type 1 DNA polymerase induces hyperphosphorylation of replication protein A and its accumulation at S-phase-specific sites of DNA damage during infection.

The treatment of mammalian cells with genotoxic substances can trigger DNA damage responses that include the hyperphosphorylation of replication protein A (RPA), a protein that plays key roles in the recognition, signaling, and repair of damaged DNA. We have previously reported that in the presence of a viral polymerase inhibitor, herpes simplex virus type 1 (HSV-1) infection induces the hyperphosphorylation of RPA (D. E. Wilkinson and S. K. Weller, J. Virol. 78:4783-4796, 2004). We initiated the present study to further characterize this genotoxic response to HSV-1 infection. Here we report that infection in the presence of polymerase inhibitors triggers an S-phase-specific response to DNA damage, as demonstrated by induction of the hyperphosphorylation of RPA and its accumulation within viral foci specific to the S phase of the cell cycle. This DNA damage response occurred in the presence of viral polymerase inhibitors and required the HSV-1 polymerase holoenzyme as well as the viral single-stranded-DNA binding protein. Treatment with an inhibitor of the viral helicase-primase did not induce the hyperphosphorylation of RPA or its accumulation in infected cells. Taken together, these results suggest that the S-phase-specific DNA damage response to infection is dependent on the specific inhibition of the polymerase. Finally, RPA hyperphosphorylation was not induced during productive infection, indicating that active viral replication does not trigger this potentially detrimental stress response.

Animals↗
PubMed2004

Werner syndrome protein, the MRE11 complex and ATR: menage-à-trois in guarding genome stability during DNA replication?

The correct execution of the DNA replication process is crucially import for the maintenance of genome integrity of the cell. Several types of sources, both endogenous and exogenous, can give rise to DNA damage leading to the DNA replication fork arrest. The processes by which replication blockage is sensed by checkpoint sensors and how the pathway leading to resolution of stalled forks is activated are still not completely understood. However, recent emerging evidence suggests that one candidate for a sensor of replication stress is ATR and that, together with a member of RecQ family helicases, Werner syndrome protein (WRN) and MRE11 complex, can collaborate to promote the restarting of DNA synthesis through the resolution of stalled replication forks. Here, we discuss how WRN, the MRE11 complex and the ATR kinase could work together in response to replication blockage to avoid DNA replication fork collapse and genome instability.

DNA Damage↗
PubMed2006

Assembly of nuclear matrix-bound protein complexes involved in non-homologous end joining is induced by inhibition of DNA topoisomerase II.

Topoisomerases maintain the DNA structure by relieving the torsional stress and alleviating other topological problems occurring in DNA during transcription and replication. Topoisomerase II appears to have a close association with the family of proteins involved in the organization of chromatin in a series of loops on the proteinaceous chromosomal matrix. Beyond its physiological functions, topoisomerase II is the target for some of the most active anticancer drugs. Inhibition of the topoisomerase II function can result in DNA double-strand breaks (DSBs) and, thus, lead to chromosomal translocations. The earliest event during DSB repair is phosphorylation of histone H2AX at S139 (so-called gammaH2AX) which is believed to serve as a focal point for the assembly of repair proteins at the DSB. In this work, we have demonstrated the formation of gammaH2AX foci in two human cell lines--K562 and HeLa--after suppression of topoisomerase II activity with etoposide. Furthermore, these foci remained visible at nuclear matrices and colocalized with the major components of non-homologous end joining (NHEJ) system of DSBs repair. Thus, inhibition of topoisomerase II activity triggers assembly of NHEJ complexes at the nuclear matrix.

Antigens, Nuclear↗
PubMed2005

Mrc1 is required for normal progression of replication forks throughout chromatin in S. cerevisiae.

Mrc1 associates with replication forks, where it transmits replication stress signals and is required for normal replisome pausing in response to nucleotide depletion. Mrc1 also plays a poorly understood role in DNA replication, which appears distinct from its role in checkpoint signaling. Here, we demonstrate that Mrc1 functions constitutively to promote normal replication fork progression. In mrc1Delta cells, replication forks proceed slowly throughout chromatin, rather than being specifically defective in pausing and progression through loci that impede fork progression. Analysis of genetic interactions with Rrm3, a DNA helicase required to resolve paused forks, indicates that Mrc1 checkpoint signaling is dispensable for the resolution of stalled replication forks and suggests that replication forks lacking Mrc1 create DNA damage that must be repaired by Rrm3. These findings elucidate a central role for Mrc1 in normal replisome function, which is distinct from its role as a checkpoint mediator, but nevertheless critical to genome stability.

Cell Cycle Proteins↗
PubMed2003

Community outreach as an iterative dialogue among scientists and communities in the Texas gulf coast region.

In response to significant environmental health challenges in Southeast Texas, a National Institute for Environmental Health Sciences Center at the University of Texas Medical Branch at Galveston was created to promote and conduct inter-disciplinary research in the areas of: (1) the molecular biology of DNA repair, replication and mutagenesis, (2) asthma pathogenesis in response to oxidative stress and viral exposures, and (3) environmental toxicant biotransformation. In addition, the NIEHS Center maintains close ties with neighboring communities through an active Community Outreach & Education Program (COEP) that develops and disseminates translational materials for use in environmental health awareness outreach, toxicology consultation, K-12 curriculum enrichment and in developing site-specific Community Partnership projects. The COEP core service divisions include: Environmental Arts & Sciences, Asthma Outreach & Education, Theater Outreach & Education, and Public Forum & Toxics Assistance. Public Forums focus on the use of Augusto Boal's Forum Theater dramaturgy to include the voices and local knowledge of communities within the process of Participatory Research. Forums create the preconditions for significant partnerships that link the hazardous risk perceptions and environmental health needs of communities with the expertise of NIEHS Center investigators and translational services provided through COEP outreach programs. The Forum process also creates leadership cores within environmentally challenged communities that facilitate the ongoing translational process and maintain the vital linkage between the health needs of communities and the analytic tools and the field and clinical technologies of the environmental sciences.

Community-Institutional Relations↗
PubMed2003

beta-Lactam induction of colanic acid gene expression in Escherichia coli.

An unexpected observation led us to examine the relationship between beta-lactam exposure and synthesis of colonic acid capsular polysaccharide in Escherichia coli. Strains containing a cps-lacZ transcriptional fusion were challenged with antibiotics having various modes of action, and gene expression was detected by a disk-diffusion assay and in broth cultures. The cps genes were induced by a subset of beta-lactams but not by agents inhibiting protein synthesis or DNA replication, indicating that cps expression was specific and not due to stresses accompanying cell death or by a general inhibition of peptidoglycan synthesis. A narrow concentration just below the MIC triggered cps expression in liquid culture, suggesting the response may be triggered by near-lethal levels of antibiotic. Because colanic acid is important for maturation of biofilm architecture, antibiotics that increase its synthesis might exacerbate the formation or persistence of biofilms.

Artificial Gene Fusion↗
PubMed2006

Ras triggers ataxia-telangiectasia-mutated and Rad-3-related activation and apoptosis through sustained mitogenic signaling.

Genetic evidence indicates that Ras plays a critical role in the initiation and progression of human thyroid tumors. Paradoxically, acute expression of activated Ras in normal rat thyroid cells induced deregulated cell cycle progression and apoptosis. We investigated whether cell cycle progression was required for Ras-stimulated apoptosis. Ras increased CDK-2 activity following its introduction into quiescent cells. Apoptotic cells exhibited a sustained increase in CDK-2 activity, accompanied by the loss of CDK-2-associated p27. Blockade of Ras-induced CDK-2 activity and S phase entry via overexpression of p27 inhibited apoptosis. Inactivation of the retinoblastoma protein in quiescent cells through expression of HPV-E7 stimulated cell cycle progression and apoptosis, indicating that deregulated cell cycle progression is sufficient to induce apoptosis. Ras failed to induce G1 phase growth arrest in normal rat thyroid cells. Rather, Ras-expressing thyroid cells progressed into S and G2 phases and evoked a checkpoint response characterized by the activation of ATR. Ras-stimulated ATR activity, as evidenced by Chk1 and p53 phosphorylation, was blocked by p27, suggesting that cell cycle progression triggers checkpoint activation, likely as a consequence of replication stress. These data reveal that Ras is capable of inducing a DNA damage response with characteristics similar to those reported in precancerous lesions. Our findings also suggest that the frequent mutational activation of Ras in thyroid tumors reflects the ability of Ras-expressing cells to bypass checkpoints and evade apoptosis rather than to simply increase proliferative potential.

Animals↗
PubMed2026

G-quadruplex structures as regulators of cellular processes and drivers of genome instability in cancer.

G-quadruplexes (G4s) are essential regulatory structures whose biological functions are inseparable from their potential to destabilize the genome. They play critical roles in transcription, replication and chromatin architecture, yet they also contribute to the genomic instability that fuels cancer. This dual role is an inherent consequence of where G4s form in the genome. G4s are enriched at highly active regulatory regions, including promoters, replication origins and topologically associated domain boundaries, where their controlled formation and resolution by helicases and topoisomerases support normal genome function. When this control is lost, the same features that make G4s functional become harmful, leading to R-loop and G-loop accumulation, replication fork stalling and increased conflicts between transcription and replication. Topoisomerase activity, which normally relieves supercoiling stress, can instead generate the DNA double-strand breaks that characterize cancer genomes. Persistent G4 structures also promote micronuclei formation and cytoplasmic DNA accumulation, activating the cGAS-STING innate immune signaling pathway. Here, we discuss these mechanisms and present pan-cancer genomic analyses showing that these processes operate broadly across human tumors. Therapeutically targeting G4s requires balancing their essential regulatory roles with their pathological effects. Understanding this tension is therefore essential for exploiting G4s as therapeutic targets across cancer types.

DEAH-box helicases↗
PubMedDate not supplied

Mechanisms of cell-cycle checkpoints: at the crossroads of carcinogenesis and drug discovery.

Human tumors arise from multiple genetic changes that gradually transform growth-limited cells into highly invasive cells that are unresponsive to growth controls. The genetic evolution of normal cells into cancer cells is largely determined by the fidelity of DNA replication, repair, and division. Cell-cycle arrest in response to stress is integral to the maintenance of genomic integrity. The control mechanisms that restrain cell-cycle transition or induce apoptotic signaling pathways after cell stress are known as cell-cycle checkpoints. This review will focus on the mechanisms of cell-cycle checkpoint pathways and how different components of these pathways are frequently altered in the genesis of human tumors. As our knowledge of cell-cycle regulation and checkpoints increases, so will our understanding of how xenobiotic agents can affect these processes to either initiate or inhibit tumorigenesis.

Antineoplastic Agents↗
PubMed2006

The fission yeast DNA structure checkpoint protein Rad26ATRIP/LCD1/UVSD accumulates in the cytoplasm following microtubule destabilization.

BACKGROUND: DNA structure checkpoints are conserved eukaryotic signal transduction pathways that help preserve genomic integrity. Upon detecting checkpoint signals such as stalled replication forks or double-stranded DNA breaks, these pathways coordinate appropriate stress responses. Members of the PI-3 kinase related kinase (PIKK) family are essential elements of DNA structure checkpoints. In fission yeast, the Rad3 PIKK and its regulatory subunit Rad26 coordinate the detection of checkpoint signals with pathway outputs. RESULTS: We found that untreated rad26Delta cells were defective for two microtubule-dependent processes: chromosome segregation and morphogenesis. Interestingly, cytoplasmic accumulation of Rad26-GFP occurred following treatment with microtubule destabilizing drugs, but not during treatment with the genotoxic agent Phleomycin. Cytoplasmic accumulation of Rad26-GFP depended on Rad24, a 14-3-3 protein also required for DNA structure checkpoints and morphogenesis. Results of over expression and epistasis experiments confirm that Rad26 and Rad24 define a response to microtubule destabilizing conditions. CONCLUSION: Two DNA structure checkpoint proteins with roles in morphogenesis define a response to microtubule destabilizing conditions.

Actins↗