Increased Genome Instability Research Articles

Strong association between cancer and genomic instability

After a first wave of radiation-induced chromosomal aberrations, a second wave appears 20-30 cell generations after radiation exposure and persists thereafter. This late effect is usually termed "genomic instability". A better term is "increased genomic instability". This effect has been observed in many cell systems in vitro and in vivo for quite a number of biological endpoints. The radiation-induced increase in genomic instability is apparently a general phenomenon. In the development of cancer, several mutations are involved. With increasing genomic instability, the probability for further mutations is enhanced. Several studies show that genomic instability is increased not only in the cancer cells but also in "normal" cells of cancer patients e.g. peripheral lymphocytes. This has for example been shown in uranium miners with bronchial carcinomas, but also in untreated head and neck cancer patients. The association between cancer and genomic instability is also found in individuals with a genetic predisposition for increased radiosensitivity. Several such syndromes have been found. In all cases, an increased genomic instability, cancer proneness and increased radiosensitivity coincide. In these syndromes, deficiencies in certain DNA-repair pathways occur as well as deregulations of the cell cycle. Especially, mutations are seen in genes encoding proteins, which are involved in the G(1)/S-phase checkpoint. Genomic instability apparently promotes cancer development. In this context, it is interesting that hypoxia, increased genomic instability and cancer are also associated. All these processes are energy dependent. Some strong evidence exists that the structure and length of telomeres is connected to the development of genomic instability.

A Systems Analysis of Mitotic Apparatus Inhibitors Defines a Response Network for Breast Cancer.

Abstract Deregulation of aspects of the mitotic apparatus leads to increased genome instability, carcinogenesis and aggressive tumor behavior in human and rodent model systems1. This knowledge has stimulated development of inhibitors of elements of the mitotic apparatus as anticancer agents including PLK1, CENPE, and AURKB and several are now being tested for efficacy clincially2-6. These trials and eventual clinical use will benefit from molecular markers that predict response. In order to identify such markers, we assessed quantitative responses to the agents GSK461364, GSK923295 and GSK1070916 that target PLK1, CENPE and AURKB; respectively, in a panel of 50 breast cancer cell lines. This analysis showed that basal subtype cell lines were preferentially sensitive to all three agents and that responses among the lines to the three agents were strongly correlated. This may be explained by our discovery that components of the mitotic apparatus including PLK1, CENPE and AURKB form a transcriptionally co-regulated network comprised of more than 50 genes that is preferentially active in basal subtype of breast cell lines and primary tumors. Remarkably, this network also is activate in subsets of cancers of the lung, ovarian, prostate and brain, Wilms tumor, human blood malignancies and selected normal tissues. We then defined a mitotic apparatus network index (MANI) and showed that high MANI was associated with poor outcome clinically and with preferential responsive to GSK461364, GSK923295 and GSK1070916 in preclinical models. This suggests that measures of the MANI will identify poor outcome tumors that will likely respond well to mitotic apparatus network gene inhibitors as well as potential dose limiting normal tissues.Reference1. Quigley, D.A. et al. Nature 458, 505-8 (2009).2. Strebhardt, K. & Ullrich, A. Nat. Rev. Cancer 6, 321-330 (2006).3. Toyoshima-Morimoto, F., Taniguchi, E., Shinya, N., Iwamatsu, A. & Nishida, E. Nature 410, 215-20 (2001).4. Barr, F.A., Sillje, H.H. & Nigg, E.A. Nat. Rev. Mol. Cell. Biol. 5, 429–440 (2004).5. McInnes, C. et al. Nat. Chem. Biol. 2, 608–617 (2006).6. Yamada, S. et al. Oncogene 23, 5901-5911(2004). Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2020.

Expression of candidate tumor suppressor gene ING2 is lost in non-small cell lung carcinoma

ING2 is a candidate tumor suppressor gene involved in cell cycle control, apoptosis and senescence. Furthermore, we have recently shown that loss of ING2 expression is associated with increased genome instability. We investigated its status in a series of 120 non-small cell lung cancer (NSCLC) by using immunohistochemistry (IHC). The results showed that ING2 protein expression is downregulated in more than 50% of NSCLC, with a higher frequency in adenocarcinoma (ADK) as compared to squamous cell carcinoma (SCC) (68% versus 45%, P = 0.021). Loss of ING2 expression occurs in a high proportion of tumors from stage I and was not associated with patient's gender, age and 5-year survival. When investigating the possible mechanisms responsible for the decrease of ING2 expression, we did not observe any loss of heterozygosity or mutation in the ING2 gene. However, in 95% of the cases examined, we identified a silent single nucleotide polymorphism (SNP). By using quantitative RT-PCR, we found that ING2 loss of expression may be due to the decrease of its mRNA level. Analysis of CpG islands present in the promoter region of the ING2 gene did not allow for the detection of methylation. Mechanistically, although p53 can regulate ING2 transcription and ING2 enhances p53 activity, no correlation between ING2 and p53 IHC status was observed. Overall, these results indicate that loss of ING2 expression could contribute to lung tumorigenesis independently of p53.

Repression of Transposable Elements by Histone Biotinylation ,

Transposable elements constitute >40% of the human genome; transposition of these elements increases genome instability and cancer risk. Epigenetic mechanisms are important for transcriptional repression of retrotransposons, thereby preventing transposition events. Binding of biotin to histones, mediated by holocarboxylase synthetase (HCS), is a novel histone mark that plays a role in gene regulation. Here, we review recent findings that biotinylation of lysine-12 in histone H4 (H4K12bio) is an epigenetic mechanism to repress long terminal repeat (LTR) retrotransposons in human and mouse cell lines, primary cells from human adults, and in Drosophila melanogaster. Further, evidence is summarized that supports a causal relationship between the repression of LTR in H4K12bio-depleted cells and increased production of viral particles, increased frequency of retrotransposition events, and increased frequency of chromosomal abnormalities in mammals and Drosophila. Although HCS interacts physically with histones H3 and H4, the mechanism responsible for targeting HCS to retrotransposons to mediate histone biotinylation is uncertain. We hypothesize that HCS binds specifically to genomic regions rich in methylated cytosines and catalyzes increased biotinylation of histone H4 at lysine-12. Further, we hypothesize that this biotinylation promotes the subsequent dimethylation of lysine-9 in histone H3, resulting in an overall synergistic effect of 3 diet-dependent covalent modifications of histones in the repression of LTR.

Hematopoietic Stem Cells Repair Ionizing Radiation-Induced DNA Double Strand Breaks in a Cell Cycle-Dependent Manner.

Abstract 3241Poster Board III-178Mice with mutations in various DNA repair genes exhibit accelerated aging due to hematopoietic stem cell (HSC) premature exhaustion, indicating that DNA repair is crucial for the maintenance of HSC self-renewal and hematopoietic function. In addition, some of these mutated mice are highly susceptible to the development of leukemia and lymphoma due to an increase in genomic instability in HSCs. However, how HSCs respond to genotoxic stress and repair DNA damage have not been well established and thus, were investigated in the present study using a mouse model. Specifically, DNA damage and repair were analyzed by gH2AX immunofluorescent staining and neutral comet assay to quantify IR-induced DNA double strand breaks (DSBs) in HSCs (Lin- c-kit+ Sca1+ cells or LKS+ cells) and hematopoietic progenitor cells (HPCs; Lin- c-kit+ Sca1- cells or LKS- cells) isolated from adult mouse bone marrow (BM) after they were exposed to ionizing radiation (IR). The results showed that exposure to IR induced a similar number of DSBs in HSCs and hematopoietic progenitor cells (HPCs) isolated from adult mouse BM. However, HPCs repaired the damage within 6 h after IR, whereas more than 50% DSBs were unrepaired by HSCs even at 24h after IR, indicating that HSCs are highly deficient in repair of IR-induced DSBs. The deficient DSBs repair in HSCs is attributable to their quiescence, as sorted quiescent Pyronin Ylow HSCs were more deficient in repairing the damage than cycling Pyronin Yhigh HSCs. This suggestion is further supported by the observations that proliferating HSCs such as fetal liver HSCs and HSCs isolated from 5-FU-treated adult mouse BM repaired the damage as efficiently as HPCs. In addition, incubation of quiescent Pyronin Ylow HSCs from adult BM with stem cell factor and thrombopoietin for 48 h stimulated the cell cycle entry and DNA damage repair function. These findings indicate that stimulation of cell cycling can promote HSCs to repair DNA damage. The difference in repair of IR-induced DSBs between quiescent and cycling HSCs is not because they express different levels of key proteins (such as Ku70, Ku80, DNA-PKcs, Lig4, XRCC4, Dclre1c, Nhej1, Brac-1, Brac-2, MRE11a, Nbs1, Rad50, Rad51, and ATM) involved in non-homologous end joining (NHEJ) and homologous recombination (HR). Instead, quiescent HSCs exhibited an insignificant activation of DNA-PK and minimal formation of XRCC4 and Rad51 foci after exposure to IR, suggesting that quiescent HSCs are deficient in DSBs repair through the NHEJ and HR pathways. However, quiescent HSCs exhibited similar levels of phosphorylation of ATM and p53 after IR compared to cycling HSCs and HPCs, indicating that quiescent HSCs are proficient in sensing DNA damage to initiate DNA damage responses. These findings provide crucial insights into how HSCs respond to and repair DNA damage, which could significantly advance our understanding on how HSCs maintain their genomic stability. DisclosuresNo relevant conflicts of interest to declare.

Deregulated Gene Expression Pathways in Myelodysplastic Syndrome Hematopoietic Stem Cells.

Abstract 735The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell malignancies that are characterized by ineffective hematopoiesis resulting in peripheral cytopenias and a hypercellular bone marrow. In order to gain insight into the molecular pathogenesis of the MDS, we have determined the transcriptome of the hematopoietic stem cells (HSC) of 183 MDS patients and 17 healthy controls. The CD34+ cells obtained from MDS patients and healthy individuals were analyzed using Affymetrix U133 Plus2.0 arrays. Global pathway analysis using the Ingenuity software and the DAVID database has identified critical deregulated gene pathways and gene ontology (functional) groups perturbed in MDS HSC compared with normal HSC. The most significantly deregulated pathways in MDS include interferon signaling, thrombopoietin signaling and the Wnt pathway. Moreover, we have identified multiple pathways that are deregulated in specific MDS karyotypic groups and between early (subtype RA) and advanced MDS (subtype RAEB2). Among the most significantly deregulated gene pathways and ontology groups in early MDS are immunodeficiency, apoptosis and chemokine signaling, whereas advanced MDS is characterized by deregulation of the cell cycle, DNA damage response and checkpoint pathways. The clinical behavior of patients with del(5q), +8 or–7/del(7q) is different and we have identified distinct gene expression profiles and deregulated gene pathways for MDS defined by these major karyotypic groups. The most significantly deregulated gene pathways in del(5q) MDS include primary immunodeficiency signaling, Wnt/beta-catenin signaling, integrin signaling, cell cycle regulation and Huntington's disease signaling. Patients with the 5q- syndrome also show deregulation of the p53 pathway. Moreover, chromatin assembly and translation are among the most significant gene ontology groups in del(5q) MDS. We have found that MDS with the–7/del(7q) is characterized by deregulation of multiple pathways involved in cell survival, differentiation, apoptosis and growth, and include SAPK/JNK, NF-kB, PI3K/AKT and ceramide signaling pathways. Strikingly, all of the most significantly deregulated gene pathways in trisomy 8 MDS in our study concern or are associated with the immune response, and include B-cell receptor signaling, antigen presentation and CTLA4 signaling in Cytotoxic T lymphocytes pathways. These data are consistent with an immune system role in the pathogenesis of MDS with trisomy 8. Importantly, much of the deregulated pathway data generated in this study is in accord with the known biology of MDS. On the basis of our observations, we suggest a model for MDS in which immune deregulation and activation of apoptosis pathways in early MDS cells, consistent with clinically observed ineffective hematopoiesis, functions as a barrier to prevent leukemic transformation. Disruption of the DNA damage check points in advanced MDS results in an increase in the error rate of DNA repair with a concomitant increase in genomic instability, leading to evolution to AML. This is the first study to determine deregulated gene pathways and ontology groups in the HSC compartment of a large group of patients with MDS. The deregulated pathways identified are likely to be critical to the MDS HSC phenotype, provide important new insights into the molecular pathogenesis of this disorder, and may represent new targets for therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

Silencing of the Lats2 tumor suppressor overrides a p53-dependent oncogenic stress checkpoint and enables mutant H-Ras-driven cell transformation

The Lats2 tumor suppressor protein has previously been implicated in promoting p53 activation in response to mitotic apparatus stress, by preventing Mdm2-driven p53 degradation. We now report that Lats2 also plays a role in an ATR-Chk1-mediated stress checkpoint in response to oncogenic H-Ras. Activated mutant H-Ras triggers the translocation of Lats2 from centrosomes into the nucleus, coupled with an increase in Lats2 protein levels. This leads to induction of p53 activity, upregulation of proapoptotic genes, downregulation of antiapoptotic genes and eventually apoptotic cell death. Many of the cells that survive apoptosis undergo senescence. However, a fraction of the cells escape this checkpoint mechanism, despite maintaining high mutant H-Ras expression. These escapers display increased genome instability, as evidenced by a substantial fraction of cells with micronuclei and cells with polyploid genomes. Interestingly, such cells exhibit markedly reduced levels of Lats2, in conjunction with enhanced hypermethylation of the Lats2 gene promoter. Our findings suggest that Lats2 might play an important role in quenching H-Ras-induced transformation, while silencing of Lats2 expression might serve as a mechanism to enable tumor progression.

Chk2 Protects against Radiation-Induced Genomic Instability

The murine Chk2 kinase is activated after exposure to ionizing radiation and is necessary for p53-dependent apoptosis, but the role Chk2 plays in determining genomic stability is poorly understood. By analyzing the sensitivity of Chk2-deficient murine and human cells to a range of DNA-damaging agents, we show that Chk2 deficiency results in resistance to agents that generate double-strand breaks but not to other forms of damage. Surprisingly, the absence of Chk2 results in increased sensitivity to UV-radiation-induced DNA damage. Defective apoptosis after radiation-induced DNA damage may result in genomic instability; therefore, the consequences of Chk2 deficiency on genomic instability were assayed using an in vitro screen. Gene amplification was not detected in untreated Chk2(-/-) cells, but the rate of gene amplification after irradiation was elevated and was similar to that found in p53 compromised cells. A synergistic increase in genomic instability was seen after disruption of both Chk2 and p53 function, indicating that the two proteins have non-redundant roles in regulating genome stability after irradiation. The data demonstrate that Chk2 functions to maintain genome integrity after radiation-induced damage and has important implications for the use of Chk2 inhibitors as adjuvant cancer therapy.

RelA/p65 functions to maintain cellular senescence by regulating genomic stability and DNA repair

Nuclear factor (NF)-kappaB is a positive regulator of tumour development and progression, but how it functions in normal cells leading to oncogenesis is not clear. As cellular senescence has proven to be an intrinsic tumour suppressor mechanism that cells must overcome to establish deregulated growth, we used primary fibroblasts to follow NF-kappaB function in cells transitioning from senescence to subsequent immortalization. Our findings show that RelA/p65(-/-) murine fibroblasts immortalize at considerably faster rates than RelA/p65(+/+) cells. The ability of RelA/p65(-/-) fibroblasts to escape senescence earlier is due to their genomic instability, characterized by high frequencies of DNA mutations, gene deletions and gross chromosomal translocations. This increase in genomic instability is closely related to a compromised DNA repair that occurs in both murine RelA/p65(-/-) fibroblasts and tissues. Significantly, these results can also be duplicated in human fibroblasts lacking NF-kappaB. Altogether, our findings present a fresh perspective on the role of NF-kappaB as a tumour suppressor, which acts in pre-neoplastic cells to maintain cellular senescence by promoting DNA repair and genomic stability.

Organophosphorous pesticides and estrogen induce transformation of breast cells affecting p53 and c-Ha-ras genes

Cancer progression has been associated with an increase in genomic instability indicated by inactivation of tumor suppressor genes and activation of oncogenes. Epidemiological and experimental evidence has implicated estrogens in the etiology of breast cancer. To study environmental organophosphorous pesticides is of interest since evidence indicate that pesticides may enhance cell division, increasing the risk of breast cancer. The aim was to evaluate the effects of these pesticides, such as parathion and malathion in the presence of estrogen on malignant transformation as well as on genomic instability, that is in the frequency of loss of heterozygosity (LOH) and microsatellite instability (MSI). The MCF-10F immortalized human breast epithelial cell line, that was treated with parathion or malathion alone and in combination with estrogen was used. These studies indicated that either pesticide alone or in combination with estrogen induced malignant transformation as shown by anchorage-independent growth capability and invasive characteristics in comparison to control. Such malignant phenotypic characteristics were corroborated by significant (P<0.05) increase in p53 and c-Ha-ras protein expression. Results indicated different degrees of allelic imbalance in the form of LOH or MSI with different microsatellite markers. MSI was found in malathion and estrogen-treated cells with a marker used for p53 tumor suppressor gene at loci 17p13.1. The same combination of substances presented MSI with a marker used for c-Ha-ras mapped in chromosome 11p14.1, as well as mutations in c-Ha-ras for codons 12 and 61. LOH was observed in codon 12 in the presence of estrogen or malathion alone. Parathion alone and combined with estrogen induced MSI in codon 61. It can be concluded that the organophosphorous pesticides parathion and malathion induced malignant transformation of breast cells through genomic instability altering p53 and c-Ha-ras, considered pivotal to cancer process.

Transgenerational changes in somatic and germ line genetic integrity of first-generation offspring derived from the DNA damaged sperm

To report a more quantitative approach to study the influence of varying levels of sperm DNA damage on transgenerational changes in genomic instability in a mouse model. Experimental prospective study. Embryology research laboratory. Swiss albino mice. The sperm DNA damage was induced by different doses of gamma-irradiation to male mice followed by mating with healthy female mice. Genomic integrity in embryos, fetus, and spermatozoa of F1 mice derived from the DNA-damaged sperm. The transgenerational changes in genetic integrity were attributed by a dose-dependent increase in the frequency of micronuclei in preimplantation embryos and a concomitant increase in genomic instability in fetal liver cells and sperm chromatin modifications in F1 males. A strong positive correlation was observed between the extent of sperm DNA damage and somatic and germ-line genomic instability. Sperm-mediated transgenerational genomic instability is dependent on the amount of DNA damage present in the sire's sperm at the time of fertilization.

Ironing Out a Midlife Crisis

There is a strong correlation between age, genomic instability, and the development of cancer. Working in yeast, Veatch et al. (2009) now propose that defects in the biogenesis of iron-sulfur clusters arising as a consequence of mitochondrial dysfunction contribute to the increase in genomic instability as cells age.

Malignant Mesothelioma: Biology, Diagnosis and Therapeutic Approaches

Malignant mesothelioma (MM) is an aggressive neoplasm of serosal cavities, which is resistant to conventional therapy, with patient survival from presentation of <12 months. MM remains a universally fatal disease of increasing incidence worldwide. Although the main risk factor is asbestos exposure, other factors, Simian virus 40 infection and inheritance of susceptibility genes, likely play a role. Asbestos-related carcinogenic process is primarily based on the interaction between susceptibility (genetic and acquired) and exposure to carcinogenic environmental agents. Asbestos-induced carcinogenesis includes generation of reactive oxygen species, which induce DNA strand breaks and oxidant-induced base modifications to DNA. Persistent oxidative DNA damage can alter signaling cascades, gene expression, induce or arrest transcription, and increase replication errors and genomic instability. The long promotion phase observed in MM pathogenesis and the absence of early symptoms both contribute to late diagnosis of the disease. This results in delayed therapeutic intervention of patients, making the outcome of the disease very grim. There have been several developments in MM management, principally based on early detection, improved diagnosis, development of more effective therapies, and new insights into the pathobiology of the disease. Several programs have been used to screen asbestos-exposed individuals for lung and pleural disease. These programs involve annual pulmonary function tests, chest radiography and high resolution computer tomography. Blood tests make screening of target populations an attractive strategy. Many current gene and protein expression studies aim to identify clinically useful biomarkers and new therapeutic targets for improved management of MM.

Malignant Mesothelioma: Biology, Diagnosis and Therapeutic Approaches

Malignant mesothelioma (MM) is an aggressive neoplasm of serosal cavities, which is resistant to conventional therapy, with patient survival from presentation of < 12 months. MM remains a universally fatal disease of increasing incidence worldwide. Although the main risk factor is asbestos exposure, other factors, Simian virus 40 infection and inheritance of susceptibility genes, likely play a role. Asbestos-related carcinogenic process is primarily based on the interaction between susceptibility (genetic and acquired) and exposure to carcinogenic environmental agents. Asbestos-induced carcinogenesis includes generation of reactive oxygen species, which induce DNA strand breaks and oxidant-induced base modifications to DNA. Persistent oxidative DNA damage can alter signaling cascades, gene expression, induce or arrest transcription, and increase replication errors and genomic instability. The long promotion phase observed in MM pathogenesis and the absence of early symptoms both contribute to late diagnosis of the disease. This results in delayed therapeutic intervention of patients, making the outcome of the disease very grim. There have been several developments in MM management, principally based on early detection, improved diagnosis, development of more effective therapies, and new insights into the pathobiology of the disease. Several programs have been used to screen asbestos-exposed individuals for lung and pleural disease. These programs involve annual pulmonary function tests, chest radiography and high resolution computer tomography. Blood tests make screening of target populations an attractive strategy. Many current gene and protein expression studies aim to identify clinically useful biomarkers and new therapeutic targets for improved management of MM. Keywords: Malignant mesothelioma, asbestos, inflammation, angiogenesis, tumorigenesis, biomarkers, diagnosis, therapy

Understanding the functions of BRCA1 in the DNA-damage response

Inheritance of a mutation in BRCA1 (breast cancer 1 early-onset) results in predisposition to early-onset breast and ovarian cancer. Tumours in these individuals arise after somatic mutation or loss of the wild-type allele. Loss of BRCA1 function leads to a profound increase in genomic instability involving the accumulation of mutations, DNA breaks and gross chromosomal rearrangements. Accordingly, BRCA1 has been implicated as an important factor involved in both the repair of DNA lesions and in the regulation of cell-cycle checkpoints in response to DNA damage. However, the molecular mechanism through which BRCA1 functions to preserve genome stability remains unclear. In the present article, we examine the different ways in which BRCA1 might influence the repair of DNA damage and the preservation of genome integrity, taking into account what is currently known about its interactions with other proteins, its biochemical activity and its nuclear localization.

SNP'ing for longevity

SNP'ing for longevity

Genomic instability and DNA damage responses in progeria arising from defective maturation of prelamin A

Progeria syndromes have in common a premature aging phenotype and increased genome instability. The susceptibility to DNA damage arises from a compromised repair system, either in the repair proteins themselves or in the DNA damage response pathways. The most severe progerias stem from mutations affecting lamin A production, a filamentous protein of the nuclear lamina. Hutchinson-Gilford progeria syndrome (HGPS) patients are heterozygous for aLMNA gene mutation while Restrictive Dermopathy (RD) individuals have a homozygous deficiency in the processing protease Zmpste24. These mutations generate the mutant lamin A proteins progerin and FC-lamina A, respectively, which cause nuclear deformations and chromatin perturbations. Genome instability is observed even though genome maintenance and repair genes appear normal. The unresolved question is what features of the DNA damage response pathways are deficient in HGPS and RD cells. Here we review and discuss recent findings which resolve some mechanistic details of how the accumulation of progerin/FC-lamin A proteins may disrupt DNA damage response pathways in HGPS and RD cells. As the mutant lamin proteins accumulate they sequester replication and repair factors, leading to stalled replication forks which collapse into DNA double-strand beaks (DSBs). In a reaction unique to HGPS and RD cells these accessible DSB termini bind Xeroderma pigmentosum group A (XPA) protein which excludes normal binding by DNA DSB repair proteins. The bound XPA also signals activation of ATM and ATR, arresting cell cycle progression, leading to arrested growth. In addition, the effective sequestration of XPA at these DSB damage sites makes HGPS and RD cells more sensitive to ultraviolet light and other mutagens normally repaired by the nucleotide excision repair pathway of which XPA is a necessary and specific component.

Telomerase Deficiency Affects the Formation of Chromosomal Translocations by Homologous Recombination in Saccharomyces cerevisiae

Telomerase is a ribonucleoprotein complex required for the replication and protection of telomeric DNA in eukaryotes. Cells lacking telomerase undergo a progressive loss of telomeric DNA that results in loss of viability and a concomitant increase in genome instability. We have used budding yeast to investigate the relationship between telomerase deficiency and the generation of chromosomal translocations, a common characteristic of cancer cells. Telomerase deficiency increased the rate of formation of spontaneous translocations by homologous recombination involving telomere proximal sequences during crisis. However, telomerase deficiency also decreased the frequency of translocation formation following multiple HO-endonuclease catalyzed DNA double-strand breaks at telomere proximal or distal sequences before, during and after crisis. This decrease correlated with a sequestration of the central homologous recombination factor, Rad52, to telomeres determined by chromatin immuno-precipitation. This suggests that telomerase deficiency results in the sequestration of Rad52 to telomeres, limiting the capacity of the cell to repair double-strand breaks throughout the genome. Increased spontaneous translocation formation in telomerase-deficient yeast cells undergoing crisis is consistent with the increased incidence of cancer in elderly humans, as the majority of our cells lack telomerase. Decreased translocation formation by recombinational repair of double-strand breaks in telomerase-deficient yeast suggests that the reemergence of telomerase expression observed in many human tumors may further stimulate genome rearrangement. Thus, telomerase may exert a substantial effect on global genome stability, which may bear significantly on the appearance and progression of cancer in humans.

The Hsk1(Cdc7) replication kinase regulates origin efficiency.

Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or decreasing Hsk1-Dfp1 levels correspondingly increases or decreases origin efficiency. Furthermore, tethering Hsk1-Dfp1 near an origin increases the efficiency of that origin, suggesting that the effective local concentration of Hsk1-Dfp1 regulates origin firing. Using photobleaching, we show that Hsk1-Dfp1 is freely diffusible in the nucleus. These results support a model in which the accessibility of replication origins to Hsk1-Dfp1 regulates origin efficiency and provides a potential mechanistic link between chromatin structure and replication timing. By manipulating Hsk1-Dfp1 levels, we show that increasing or decreasing origin firing rates leads to an increase in genomic instability, demonstrating the biological importance of appropriate origin efficiency.

Role of DNA Mismatch Repair and Double-Strand Break Repair in Genome Stability and Antifungal Drug Resistance in Candida albicans

Drug resistance has become a major problem in the treatment of Candida albicans infections. Genome changes, such as aneuploidy, translocations, loss of heterozygosity, or point mutations, are often observed in clinical isolates that have become resistant to antifungal drugs. To determine whether these types of alterations result when DNA repair pathways are eliminated, we constructed yeast strains bearing deletions in six genes involved in mismatch repair (MSH2 and PMS1) or double-strand break repair (MRE11, RAD50, RAD52, and YKU80). We show that the mre11Delta/mre11Delta, rad50Delta/rad50Delta, and rad52Delta/rad52Delta mutants are slow growing and exhibit a wrinkly colony phenotype and that cultures of these mutants contain abundant elongated pseudohypha-like cells. These same mutants are susceptible to hydrogen peroxide, tetrabutyl hydrogen peroxide, UV radiation, camptothecin, ethylmethane sulfonate, and methylmethane sulfonate. The msh2Delta/msh2Delta, pms1Delta/pms1Delta, and yku80Delta/yku80Delta mutants exhibit none of these phenotypes. We observed an increase in genome instability in mre11Delta/mre11Delta and rad50Delta/rad50Delta mutants by using a GAL1/URA3 marker system to monitor the integrity of chromosome 1. We investigated the acquisition of drug resistance in the DNA repair mutants and found that deletion of mre11Delta/mre11Delta, rad50Delta/rad50Delta, or rad52Delta/rad52Delta leads to an increased susceptibility to fluconazole. Interestingly, we also observed an elevated frequency of appearance of drug-resistant colonies for both msh2Delta/msh2Delta and pms1Delta/pms1Delta (MMR mutants) and rad50Delta/rad50Delta (DSBR mutant). Our data demonstrate that defects in double-strand break repair lead to an increase in genome instability, while drug resistance arises more rapidly in C. albicans strains lacking mismatch repair proteins or proteins central to double-strand break repair.