Mechanism of Cytotoxicity of Anticancer Platinum Drugs: Evidence That Cis-diamminedichloroplatinum(II) and Cis-diammine-(1,1-cyclobutanedicarboxylato)platinum(II) Differ Only in the Kinetics of Their Interaction with DNA

Cancer Research. Apr, 1986  |  Pubmed ID: 3512077

The kinetics of the aquation reactions of cisplatin and carboplatin and their subsequent reactions with DNA, both in vitro and in vivo, have been measured. The results have been extrapolated to indicate the expected cytotoxicity of these compounds in cells obtained from human cancer patients. Rate constants for the aquation at 37 degrees C of cisplatin and carboplatin of 8 X 10(-5) and 7.2 X 10(-7) s-1, respectively, were calculated from the half-life of these compounds in phosphate buffer, pH 7. This difference in their rate of activation was matched by their rates of binding to DNA. By use of a 14C-labeled ligand, carboplatin was shown to bind monofunctionally to DNA, after which there was a time-dependent formation of difunctional interstrand cross-links, formed from some of these initially monofunctional adducts. A similar, although faster, accumulation of cross-links was seen when cisplatin was bound to DNA. The loss of the 14C-CBDCA ligand of carboplatin was calculated to occur with a rate constant of 1.3 X 10(-5) s-1 which was similar to that for the rate of formation of interstrand cross-links and faster than that for the monofunctional reaction with DNA. It was concluded therefore that the CBDCA ligand becomes a more labile leaving group once carboplatin has been monoaquated. In contrast, both chloro-ligands of cisplatin were shown to leave at similar rates. The fact that other difunctional lesions were formed to the same extent, by equal bound doses of cisplatin or carboplatin, was indicated by the unwinding of supercoiled plasmid DNA. The effects of cisplatin and carboplatin on this DNA were the same once bound to the same extent. About a 100-fold larger dose of carboplatin was, as predicted by their rates of aquation, required to produce equivalent binding to plasmid DNA. In vivo, equal binding of the two drugs to DNA of various cell systems resulted in equal cytotoxicity. Again a much larger dose (20- to 40-fold) of carboplatin was required to produce this equal binding. In general a DNA bound platinum level of about 20 nmol/g reduced cell survival by 90%, although certain cell lines were shown to be much more sensitive to DNA bound platinum. Similar binding values, to those above, were obtained in the DNA extracted from cells of human cancer patients treated with cisplatin. It was inferred that the cytotoxic effect of this level of platinum on DNA would be (unless the cells were of a sensitive phenotype) about 90%.(ABSTRACT TRUNCATED AT 400 WORDS)

The Effect of Monofunctional or Difunctional Platinum Adducts and of Various Other Associated DNA Damage on the Expression of Transfected DNA in Mammalian Cell Lines Sensitive or Resistant to Difunctional Agents

Biochimica Et Biophysica Acta. Apr, 1987  |  Pubmed ID: 3567197

The effects of introducing various DNA damage into pSV2gpt DNA on the subsequent expression of xanthine guanine phosphoribosyltransferase (XGPRT), after its transfection into two Walker 256 cell lines, one which is inherently sensitive only to difunctional agents while the other shows a normal sensitivity, have been examined. Both the sensitive (WS) and the relatively resistant (WR) cell lines were shown to be equally capable of both ligation of DNA double-strand breaks (although the efficiency varied with the actual site of the break) introduced into pSV2gpt and homologous recombination of pSV2gpt fragments (recombination events are thought to be important in the repair of DNA-DNA interstrand crosslinks). Reacting the plasmid with either the difunctional platinum compound, Cisplatin, or the monofunctional reacting Pt(Dien) caused a dose-dependent decrease in the subsequent expression of XGPRT. This decrease was about the same with either agent in either cell line when expressed as a function of dose of drug. However, when the actual binding of platinum to DNA by these compounds was measured, a large difference (due to the higher specific binding of Pt(Dien) to DNA) in the effects of the difunctional, as opposed to the monofunctional agent, was apparent and this was a reflection of the relative cytotoxicities of these compounds towards mammalian cells. Although at doses of Cisplatin equitoxic to WS and WR cells 20-fold less Pt is bound to the DNA of WS cells, no significant difference was seen on the expression of pSV2gpt, reacted with this agent, between WS or WR cells. Based upon a knowledge of the proportions of adducts formed in DNA reacted with Cisplatin, the lesion that inactivates expression of XGPRT was probably the intrastrand crosslink and it was calculated that due to the size of the plasmid, the interstrand crosslink was unlikely to be present at these inactivating doses. It is suggested that the inherent sensitivity of WS cells only to difunctional agents is due to their response to such relatively rare lesions such as a DNA-DNA interstrand crosslink.

The Identification of a Second Cell Cycle Control on the HO Promoter in Yeast: Cell Cycle Regulation of SW15 Nuclear Entry

Cell. Aug, 1990  |  Pubmed ID: 2167175

HO encodes a site-specific endonuclease that initiates mating type switching in S. cerevisiae. It is expressed only transiently during the cell cycle of mother cells, as they undergo Start, but not in daughter cells. Since SWI5 appears to be the only HO transcription factor missing when daughter cells undergo Start, we were interested in the intracellular distribution of SWI5 at cell division. We discovered that SWI5 is found equally concentrated in the nuclei of both mother and daughter cells at the end of anaphase, suggesting that its subsequent fate must somehow differ. Prior to the end of anaphase, SWI5 accumulates in the cytoplasm and only moves into the nucleus when cells enter G1. A version of the HO promoter that has lost its dependence on Start is nevertheless still strongly cell cycle regulated and is activated when SWI5 moves into the nucleus.

The Walker 256 Carcinoma: a Cell Type Inherently Sensitive Only to Those Difunctional Agents That Can Form DNA Interstrand Crosslinks

Mutation Research. Nov, 1991  |  Pubmed ID: 1719394

The Walker 256 rat tumour has been maintained in vivo for over 60 years and until recently was used as a primary screen for new antitumour agents. This screen was particularly useful in identifying difunctional alkylating agents as potentially useful anticancer agents and it would seem that the Walker tumour is composed of cells sensitive towards this type of agent. A cell line (WS) established from the Walker tumour retained the sensitivity of the tumour towards difunctional agents and we have examined its phenotype in comparison to a derived, resistant, cell line (WR). The response of WR cells to a range of cytotoxic agents was similar to other established cell lines whilst WS cells were much more sensitive only towards difunctional reacting agents. There were no significant differences in the binding of these agents to the DNA of WS or WR cells. All the agents towards which WS cells showed sensitivity were, without exception, capable of reacting with DNA in Walker cells and forming DNA-DNA interstrand crosslinks. WS cells were not sensitive to busulphan, BCNU, CCNU or Me-CCNU but these agents did not produce interstrand crosslinks in the DNA of either WS or WR cells. Thus WS cells are intrinsically sensitive to specific DNA damage and this is probably a DNA interstrand crosslink. Hybrid cells produced by fusion of WS with WR cells lacked the inherent sensitivity of the WS cells towards cisplatin; sensitivity was therefore a recessive characteristic. Transfection of WS cells with human DNA also gave rise to 2 cisplatin-resistant clones, although it could not be ascertained if these clones were true transfectants or revertants. The survival of these resistant clones, after treatment with cisplatin, was about the same as WR cells a finding which would be consistent with complementation by a transferred gene or reversion of a single gene defect in WS cells. In their sensitivity only to difunctional compounds and lack of an apparent DNA excision repair defect the phenotype of Walker cells strongly resembles those cells from human patients suffering from Fanconi's anaemia and also of yeast snm1 mutant cells. The mechanisms giving rise to this failure to tolerate specific DNA damage (which seems to involve the inability to recover from the initial inhibition of DNA synthesis and may involve a single defect of a gene involved in the late steps of crosslink repair), do not involve drug uptake, drug binding to DNA, cell size, cell doubling time or DNA excision repair.

A New Role for MCM1 in Yeast: Cell Cycle Regulation of SW15 Transcription

Genes & Development. Dec, 1991  |  Pubmed ID: 1752436

In the yeast Saccharomyces cerevisiae cell cycle-regulated SW15 transcription is essential for ensuring that mother and not daughter cells switch mating type. We have identified a 55-bp promoter sequence that appears to be responsible for restricting transcription to the late S, G2, and M phases of the cell cycle. Two proteins, MCM1, a transcription factor described previously, and SFF (SWI five factor, a newly identified factor) bind this sequence in vitro. MCM1 binds the DNA tightly on its own, but SFF will only bind as part of a ternary complex with MCM1. We observe a strong correlation between the ability of mutated SWI5 promoter sequences to form a ternary MCM1-SFF-containing complex in vitro and to activate transcription in vivo, which suggests that efficient transcription requires that both proteins bind DNA. Through its interactions with cell type-specific coactivators and corepressors, MCM1 controls cell type-specific expression of pheromone and receptor genes. By analogy, we propose that SFF enables MCM1 to function as a part of a cell cycle-regulated transcription complex.

Changes in a SWI4,6-DNA-binding Complex Occur at the Time of HO Gene Activation in Yeast

Genes & Development. Nov, 1991  |  Pubmed ID: 1936990

The yeast HO gene is transcribed transiently during G1 as cells undergo START. START-specific HO activation requires two proteins, SWI4 and SWI6, which act via a motif (CACGA4) repeated up to 10 times within the URS2 region of the HO promoter. We identified a DNA-binding activity containing SWI4 and SWI6 that recognizes the CACGA4 sequences within URS2. Two forms of SWI4,6-DNA complexes called L and U can be distinguished by their electrophoretic mobility. L complexes can be detected at all stages of the cell cycle, but U complexes are only detected in cells that have undergone START. The formation of U complexes may be the trigger of HO activation. The SWI6 protein is concentrated in the nucleus throughout G1, but at some point in S or G2 significant amounts accumulate in the cytoplasm. This change in cellular location of the SWI6 protein might contribute to the turnoff of HO transcription after cells have undergone START.

Cell Cycle Checkpoints, Genetic Instability and Cancer

Seminars in Cancer Biology. Apr, 1993  |  Pubmed ID: 8513148

During the cell cycle, the order of events is maintained by controls termed checkpoints. Two checkpoints are sensitive to DNA damage, one that acts before mitosis and a second that acts before DNA replication. This is relevant to cancer because checkpoint mutants show genetic instability, and such instability is characteristic of many cancers. Studies of checkpoints in normal and cancer cells suggest a mechanistic relationship to the central cell cycle control p34CDC2 and its regulators. We suggest how mutations in these genes and those with a role in DNA metabolism may affect the function of checkpoints. A further link between checkpoints and cancer may be the p53 protein, which appears to function at the G1-S checkpoint. Consideration of checkpoints may provide more effective means for cancer treatment.

Yeast Checkpoint Genes in DNA Damage Processing: Implications for Repair and Arrest

Science (New York, N.Y.). Dec, 1995  |  Pubmed ID: 7491494

Yeast checkpoint control genes were found to affect processing of DNA damage as well as cell cycle arrest. An assay that measures DNA damage processing in vivo showed that the checkpoint genes RAD17, RAD24, and MEC3 activated an exonuclease that degrades DNA. The degradation is probably a direct consequence of checkpoint protein function, because RAD17 encodes a putative 3'-5' DNA exonuclease. Another checkpoint gene, RAD9, had a different role: It inhibited the degradation by RAD17, RAD24, and MEC3. A model of how processing of DNA damage may be linked to both DNA repair and cell cycle arrest is proposed.

From DNA Damage to Cell Cycle Arrest and Suicide: a Budding Yeast Perspective

Current Opinion in Genetics & Development. Feb, 1996  |  Pubmed ID: 8791492

Eukaryotic checkpoint control genes are important for cell cycle delay, DNA repair and cell suicide after DNA is damaged. Recent studies in budding yeast show how the participation of checkpoint control proteins in DNA metabolism could lead to all three of these outcomes.

A Meiotic Recombination Checkpoint Controlled by Mitotic Checkpoint Genes

Nature. Oct, 1996  |  Pubmed ID: 8893012

In budding yeast, meiotic recombination occurs at about 200 sites per cell and involves DNA double-strand break (DSB) intermediates. Here we provide evidence that a checkpoint control requiring the mitotic DNA-damage checkpoint genes RAD17, RAD24 and MEC1 ensures that meiotic recombination is complete before the first meiotic division (MI). First, RAD17, RAD24 and MEC1 are required for the meiotic arrest caused by blocking the repair of DSBs with a mutation in the recA homologue DMC1. Second, mec1 and rad24 single mutants (DMC1+) appear to undergo MI before all recombination events are complete. Curiously, the mitosis-specific checkpoint gene RAD9 is not required for meiotic arrest of dmc1 mutants. This shows that although mitotic and meiotic control mechanisms are related, they differ significantly. Rad17 and Rad24 proteins may contribute directly to formation of an arrest signal by association with single-strand DNA in mitosis and meiosis.

Use of Cdc13-1-induced DNA Damage to Study Effects of Checkpoint Genes on DNA Damage Processing

Methods in Enzymology. 1997  |  Pubmed ID: 9251038

G2/M Checkpoint Genes of Saccharomyces Cerevisiae: Further Evidence for Roles in DNA Replication And/or Repair

Molecular & General Genetics : MGG. Nov, 1997  |  Pubmed ID: 9435789

We have cloned, sequenced and disrupted the checkpoint genes RAD17, RAD24 and MEC3 of Saccharomyces cerevisiae. Mec3p shows no strong similarity to other proteins currently in the database. Rad17p is similar to Rec1 from Ustilago maydis, a 3' to 5' DNA exonuclease/checkpoint protein, and the checkpoint protein Rad1p from Schizosaccharomyces pombe (as we previously reported). Rad24p shows sequence similarity to replication factor C (RFC) subunits, and the S. pombe Rad17p checkpoint protein, suggesting it has a role in DNA replication and/or repair. This hypothesis is supported by our genetic experiments which show that overexpression of RAD24 strongly reduces the growth rate of yeast strains that are defective in the DNA replication/repair proteins Rfc1p (cdc44), DNA pol alpha (cdc17) and DNA pol delta (cdc2) but has much weaker effects on cdc6, cdc9, cdc15 and CDC4 strains. The idea that RAD24 overexpression induces DNA damage, perhaps by interfering with replication/repair complexes, is further supported by our observation that RAD24 overexpression increases mitotic chromosome recombination in CDC4 strains. Although RAD17, RAD24 and MEC3 are not required for cell cycle arrest when S phase is inhibited by hydroxyurea (HU), they do contribute to the viability of yeast cells grown in the presence of HU, possibly because they are required for the repair of HU-induced DNA damage. In addition, all three are required for the rapid death of cdc13 rad9 mutants. All our data are consistent with models in which RAD17, RAD24 and MEC3 are coordinately required for the activity of one or more DNA repair pathways that link DNA damage to cell cycle arrest.

NDD1, a High-dosage Suppressor of Cdc28-1N, is Essential for Expression of a Subset of Late-S-phase-specific Genes in Saccharomyces Cerevisiae

Molecular and Cellular Biology. May, 1999  |  Pubmed ID: 10207056

cdc28-1N mutants progress through the G1 and S phases normally at the restrictive temperature but fail to undergo nuclear division. We have isolated a gene, NDD1, which at a high dosage suppresses the nuclear-division defect of cdc28-1N. NDD1 (nuclear division defective) is an essential gene. Its expression during the cell cycle is tightly regulated such that NDD1 RNA is most abundant during the S phase. Cells lacking the NDD1 gene arrest with an elongated bud, a short mitotic spindle, 2N DNA content, and an undivided nucleus, suggesting that its function is required for some aspect of nuclear division. We show that overexpression of Ndd1 results in the upregulation of both CLB1 and CLB2 transcription, suggesting that the suppression of cdc28-1N by NDD1 may be due to an accumulation of these cyclins. Overproduction of Ndd1 also enhances the expression of SWI5, whose transcription, like that of CLB1 and CLB2, is activated in the late S phase. Ndd1 is essential for the expression of CLB1, CLB2, and SWI5, since none of these genes are transcribed in its absence. Both CLB2 expression and its upregulation by NDD1 are mediated by a 240-bp promoter sequence that contains four MCM1-binding sites. However, Ndd1 does not appear to be a component of any of the protein complexes assembled on this DNA fragment, as indicated by gel mobility shift assays. Instead, overexpression of NDD1 prevents the formation of one of the complexes whose appearance correlates with the termination of CLB2 expression in G1. The inability of GAL1 promoter-driven CLB2 to suppress the lethality of NDD1 null mutant suggests that, in addition to CLB1 and CLB2, NDD1 may also be required for the transcription of other genes whose functions are necessary for G2/M transition.

Quantitative Amplification of Single-stranded DNA (QAOS) Demonstrates That Cdc13-1 Mutants Generate SsDNA in a Telomere to Centromere Direction

Nucleic Acids Research. Nov, 2001  |  Pubmed ID: 11691929

We have developed a method that allows quantitative amplification of single-stranded DNA (QAOS) in a sample that is primarily double-stranded DNA (dsDNA). Single-stranded DNA (ssDNA) is first captured by annealing a tagging primer at low temperature. Primer extension follows to create a novel, ssDNA-dependent, tagged molecule that can be detected by PCR. Using QAOS levels of between 0.2 and 100% ssDNA can be accurately quantified. We have used QAOS to characterise ssDNA levels at three loci near the right telomere of chromosome V in budding yeast cdc13-1 mutants. Our results confirm and extend previous studies which demonstrate that when Cdc13p, a telomere-binding protein, is disabled, loci close to the telomere become single stranded whereas centromere proximal sequences do not. In contrast to an earlier model, our new results are consistent with a model in which a RAD24-dependent, 5' to 3' exonuclease moves from the telomere toward the centromere in cdc13-1 mutants. QAOS has been adapted, using degenerate tagging primers, to preferentially amplify all ssDNA sequences within samples that are primarily dsDNA. This approach may be useful for identifying ssDNA sequences associated with physiological or pathological states in other organisms.

EXO1-dependent Single-stranded DNA at Telomeres Activates Subsets of DNA Damage and Spindle Checkpoint Pathways in Budding Yeast Yku70Delta Mutants

Genes & Development. Aug, 2002  |  Pubmed ID: 12154123

We have examined the role of checkpoint pathways in responding to a yku70Delta defect in budding yeast. We show that CHK1, MEC1, and RAD9 checkpoint genes are required for efficient cell cycle arrest of yku70Delta mutants cultured at 37 degrees C, whereas RAD17, RAD24, MEC3, DDC1, and DUN1 play insignificant roles. We establish that cell cycle arrest of yku70Delta mutants is associated with increasing levels of single-stranded DNA in subtelomeric Y' regions, and find that the mismatch repair-associated EXO1 gene is required for both ssDNA generation and cell cycle arrest of yku70Delta mutants. In contrast, MRE11 is not required for ssDNA generation. The behavior of yku70Delta exo1Delta double mutants strongly indicates that ssDNA is an important component of the arrest signal in yku70Delta mutants and demonstrates a link between damaged telomeres and mismatch repair-associated exonucleases. This link is confirmed by our demonstration that EXO1 also plays a role in ssDNA generation in cdc13-1 mutants. We have also found that the MAD2 but not the BUB2 spindle checkpoint gene is required for efficient arrest of yku70Delta mutants. Therefore, subsets of both DNA-damage and spindle checkpoint pathways cooperate to regulate cell division of yku70Delta mutants.

Hiding at the Ends of Yeast Chromosomes: Telomeres, Nucleases and Checkpoint Pathways

Journal of Cell Science. Oct, 2003  |  Pubmed ID: 12972499

Telomeres stabilise DNA at the ends of chromosomes, preventing chromosome fusion and genetic instability. Telomeres differ from double strand breaks in that they activate neither DNA repair nor DNA damage checkpoint pathways. Paradoxically DNA repair and checkpoint genes play critical roles in telomere stability. Recent work has provided insights into the roles of DNA repair and DNA damage checkpoint pathways in the physiological maintenance of telomeres and in cellular responses when telomeres become uncapped. In budding yeast the Mre11p nuclease, along with other unidentified nucleases, plays critical roles in physiological telomere maintenance. However, when telomeres are uncapped, the 5'-to-3' exonuclease, Exo1p, plays a critical role in generating single-stranded DNA and activating checkpoint pathways. Intriguingly Exo1p does not play an important role in normal telomere maintenance. Although checkpoint pathways are not normally activated by telomeres, at least four different types of telomere defect activate checkpoint pathways. Interestingly, each of these telomere defects depends on a different subset of checkpoint proteins to induce cell cycle arrest. A model for how a spectrum of telomeric states might interact with telomerase and checkpoint pathways is proposed.

A Domain of Rad9 Specifically Required for Activation of Chk1 in Budding Yeast

Journal of Cell Science. Feb, 2004  |  Pubmed ID: 14709724

The Rad9 protein is a key adaptor protein in Saccharomyces cerevisiae DNA damage checkpoint pathways. Its adaptor function is to link the activity of the Mec1 kinase to the activation of two parallel signalling pathways dependent on the Rad53 and Chk1 kinases. The mechanisms by which Rad9 interacts with, and activates, Rad53 are well understood. However, little was known about how Rad9 facilitates the activation of Chk1. We show here that the N-terminus of Rad9 is specifically important for phosphorylation and activation of the Chk1 kinase but not for the phosphorylation and activation of the Rad53 kinase. The Chk1 activation domain (CAD) of Rad9 is specifically important for signalling cell-cycle arrest after cdc13-1- and yku70Delta-induced telomere damage but not for tolerating ultraviolet-induced damage or inhibiting nuclease activity at telomeres. This work extends data showing that separable domains within the Rad9 adaptor protein allow it to activate two distinct kinase signalling pathways independently of each other.

Mec1 and Rad53 Inhibit Formation of Single-stranded DNA at Telomeres of Saccharomyces Cerevisiae Cdc13-1 Mutants

Genetics. Feb, 2004  |  Pubmed ID: 15020465

Here we examine the roles of budding-yeast checkpoint proteins in regulating degradation of dsDNA to ssDNA at unprotected telomeres (in Cdc13 telomere-binding protein defective strains). We find that Rad17, Mec3, as well as Rad24, members of the putative checkpoint clamp loader (Rad24) and sliding clamp (Rad17, Mec3) complexes, are important for promoting degradation of dsDNA in and near telomere repeats. We find that Mec1, Rad53, as well as Rad9, have the opposite role: they inhibit degradation. Downstream checkpoint kinases Chk1 and Dun1 play no detectable role in either promoting degradation or inhibiting it. These data suggest, first, that the checkpoint sliding clamp regulates and/or recruits some nucleases for degradation, and, second, that Mec1 activates Rad9 to activate Rad53 to inhibit degradation. Further analysis shows that Rad9 inhibits ssDNA generation by both Mec1/Rad53-dependent and -independent pathways. Exo1 appears to be targeted by the Mec1/Rad53-dependent pathway. Finally, analysis of double mutants suggests a minor role for Mec1 in promoting Rad24-dependent degradation of dsDNA. Thus, checkpoint proteins orchestrate carefully ssDNA production at unprotected telomeres.

EXO1 Plays a Role in Generating Type I and Type II Survivors in Budding Yeast

Genetics. Apr, 2004  |  Pubmed ID: 15126386

Telomerase-defective budding yeast cells escape senescence by using homologous recombination to amplify telomeric or subtelomeric structures. Similarly, human cells that enter senescence can use homologous recombination for telomere maintenance, when telomerase cannot be activated. Although recombination proteins required to generate telomerase-independent survivors have been intensively studied, little is known about the nucleases that generate the substrates for recombination. Here we demonstrate that the Exo1 exonuclease is an initiator of the recombination process that allows cells to escape senescence and become immortal in the absence of telomerase. We show that EXO1 is important for generating type I survivors in yku70delta mre11delta cells and type II survivors in tlc1delta cells. Moreover, in tlc1delta cells, EXO1 seems to contribute to the senescence process itself.

Exo1 and Rad24 Differentially Regulate Generation of SsDNA at Telomeres of Saccharomyces Cerevisiae Cdc13-1 Mutants

Genetics. Sep, 2004  |  Pubmed ID: 15454530

Cell cycle arrest in response to DNA damage depends upon coordinated interactions between DNA repair and checkpoint pathways. Here we examine the role of DNA repair and checkpoint genes in responding to unprotected telomeres in budding yeast cdc13-1 mutants. We show that Exo1 is unique among the repair genes tested because like Rad9 and Rad24 checkpoint proteins, Exo1 inhibits the growth of cdc13-1 mutants at the semipermissive temperatures. In contrast Mre11, Rad50, Xrs2, and Rad27 contribute to the vitality of cdc13-1 strains grown at permissive temperatures, while Din7, Msh2, Nuc1, Rad2, Rad52, and Yen1 show no effect. Exo1 is not required for cell cycle arrest of cdc13-1 mutants at 36 degrees but is required to maintain arrest. Exo1 affects but is not essential for the production of ssDNA in subtelomeric Y' repeats of cdc13-1 mutants. However, Exo1 is critical for generating ssDNA in subtelomeric X repeats and internal single-copy sequences. Surprisingly, and in contrast to Rad24, Exo1 is not essential to generate ssDNA in X or single-copy sequences in cdc13-1 rad9Delta mutants. We conclude that Rad24 and Exo1 regulate nucleases with different properties at uncapped telomeres and propose a model to explain our findings.

Telomerase- and Recombination-independent Immortalization of Budding Yeast

Genes & Development. Nov, 2004  |  Pubmed ID: 15489288

It is generally assumed that there are only two ways to maintain the ends of chromosomes in yeast and mammalian nuclei: telomerase and recombination. Without telomerase and recombination, cells enter senescence, a state of permanent growth arrest. We found that the decisive role in preventing senescent budding yeast cells from dividing is played by the Exo1 nuclease. In the absence of Exo1, telomerase- and recombination-defective yeast can resume cell cycle progression, despite degradation of telomeric regions from many chromosomes. As degradation progresses toward internal chromosomal regions, a progressive decrease in viability would be expected, caused by loss of essential genes. However, this was not the case. We demonstrate that extensive degradation and loss of essential genes can be efficiently prevented through a little-studied mechanism of DNA double-strand-break repair, in which short DNA palindromes induce formation of large DNA palindromes. For the first time, we show that large palindromes form as a natural consequence of postsenescence growth and that they become essential for immortalization in the absence of telomerase activity.

The PAL-mechanism of Chromosome Maintenance: Causes and Consequences

Cell Cycle (Georgetown, Tex.). Jun, 2005  |  Pubmed ID: 15970690

It is generally accepted that cells with extensive, un-repaired DNA damage can escape cell cycle arrest only by disabling checkpoint pathways and they usually perish, after several divisions, presumably due to catastrophic events on their chromosomes. Our recently discovered PAL-mechanism opens a new perspective, that some eukaryotic cells with short chromosome ends (telomeres), usually detected as DNA damage, can escape permanent cell cycle arrest (senescence) under special conditions, despite having intact checkpoints and even immortalize, despite lacking telomerase or other telomere elongation mechanisms. Here we present the first evidence that telomerase-lacking, senescent cells generate DNA damage (single stranded DNA) at internal chromosomal regions, while the telomere proximal single stranded DNA appears to be either lost or repaired. This first evidence is from the budding yeast model system. We also discuss the possible involvement of the PAL-mechanism in carcinogenesis.

Chromatin and the DNA Damage Response

DNA Repair. Sep, 2005  |  Pubmed ID: 16046284

The impact of chromatin structure upon the DNA damage response is becoming increasingly apparent. We can reasonably expect many more papers showing how chromatin and chromatin modifications impact upon aspects of the DNA damage response. Here, we present our perspective on some recent developments in this exciting area of cell biology. We aim that this review will be of interest to those who study the DNA damage response, but not usually in the context of chromatin, and equally to those who study chromatin, but not the DNA damage response. It seems likely that these two communities will increasingly share common questions and interests.

Pulsed-field Gel Electrophoresis of Budding Yeast Chromosomes

Methods in Molecular Biology (Clifton, N.J.). 2006  |  Pubmed ID: 16118425

Pulsed-field gel electrophoresis (PFGE) can be used to separate the 16 budding yeast chromosomes on the basis of size. Here we describe a detailed, practical protocol that will allow a novice to perform informative PFGE experiments. We first describe the culture of yeast prior to analysis, along with details of embedding cells in agarose before removal of cell walls. We then detail the procedure to remove protein and RNA from chromosomes and how naked chromosomes are loaded into agarose gels before being subjected to electrophoresis. Finally, we describe how the separated chromosomes can be visualized and photographed.

A Genome-wide Screen Identifies the Evolutionarily Conserved KEOPS Complex As a Telomere Regulator

Cell. Mar, 2006  |  Pubmed ID: 16564010

Telomere capping is the essential function of telomeres. To identify new genes involved in telomere capping, we carried out a genome-wide screen in Saccharomyces cerevisiae for suppressors of cdc13-1, an allele of the telomere-capping protein Cdc13. We report the identification of five novel suppressors, including the previously uncharacterized gene YML036W, which we name CGI121. Cgi121 is part of a conserved protein complex -- the KEOPS complex -- containing the protein kinase Bud32, the putative peptidase Kae1, and the uncharacterized protein Gon7. Deletion of CGI121 suppresses cdc13-1 via the dramatic reduction in ssDNA levels that accumulate in cdc13-1 cgi121 mutants. Deletion of BUD32 or other KEOPS components leads to short telomeres and a failure to add telomeres de novo to DNA double-strand breaks. Our results therefore indicate that the KEOPS complex promotes both telomere uncapping and telomere elongation.

Positional Cloning of a Cyromazine Resistance Gene in Drosophila Melanogaster

Insect Molecular Biology. Apr, 2006  |  Pubmed ID: 16640728

Cyromazine is an effective insecticide used to control dipteran insects. Its precise mode of action is yet to be determined, although it has been suggested that it interferes with the hormone system, sclerotization of the cuticle, or nucleic acid metabolism. To understand the way in which cyromazine acts, we have positionally cloned a cyromazine resistance gene from Drosophila melanogaster. Six cyromazine resistance alleles had previously been generated by ethyl methanasulphonate treatment. Two of these failed to complement each other and here we identify them as having independent non-sense mutations in CG32743, which is an ortholog of Smg1 of worms and mammals and encodes a phosphatidylinositol kinase-like kinase (PIKK). RNAi experiments confirm that cyromazine resistance can be achieved by knocking down CG32743. These are the first cyromazine resistant mutations identified at the nucleotide level. In mammals Smg1 phosphorylates P53 in response to DNA damage. This finding supports the hypothesis that cyromazine interferes with nucleic acid metabolism.

MRX Protects Telomeric DNA at Uncapped Telomeres of Budding Yeast Cdc13-1 Mutants

DNA Repair. Jul, 2006  |  Pubmed ID: 16765654

MRX, an evolutionally conserved DNA damage response complex composed of Mre11, Rad50 and Xrs2, is involved in DNA double strand break (DSB) repair, checkpoint activation and telomere maintenance. At DSBs, MRX plays a role in generating single stranded DNA (ssDNA) and signalling cell cycle arrest. Here we investigated whether MRX also contributes to generating ssDNA or signalling cell cycle arrest at uncapped telomeres. To investigate the role of MRX, we generated a conditionally degradable Rad50 protein and combined this with cdc13-1, a temperature sensitive mutation in the Cdc13 telomere capping protein. We show that Rad50 does not contribute to ssDNA generation or cell cycle arrest in response to cdcl3-1 uncapped telomeres. Instead, we find that Rad50 inhibits ssDNA accumulation and promotes cdc13-1 cell viability, consistent with a major role for MRX in telomere capping.

Linear Chromosome Maintenance in the Absence of Essential Telomere-capping Proteins

Nature Cell Biology. Jul, 2006  |  Pubmed ID: 16767084

Telomeres were defined by their ability to cap chromosome ends. Proteins with high affinity for the structure at chromosome ends, binding the G-rich, 3' single-stranded overhang at telomeres include Pot1 in humans and fission yeast, TEBP in Oxytricha nova and Cdc13 in budding yeast. Cdc13 is considered essential for telomere capping because budding yeast that lack Cdc13 rapidly accumulate excessive single-stranded DNA (ssDNA) at telomeres, arrest cell division and die. Cdc13 has a separate, critical role in telomerase recruitment to telomeres. Here, we show that neither Cdc13 nor its partner Stn1 are necessary for telomere capping if nuclease activities that are active at uncapped telomeres are attenuated. Recombination-dependent and -independent mechanisms permit maintenance of chromosomes without Cdc13. Our results indicate that the structure of the eukaryotic telomere cap is remarkably flexible and that changes in the DNA damage response allow alternative strategies for telomere capping to evolve.

Detecting Repair Intermediates in Vivo: Effects of DNA Damage Response Genes on Single-stranded DNA Accumulation at Uncapped Telomeres in Budding Yeast

Methods in Enzymology. 2006  |  Pubmed ID: 16793407

Single-stranded DNA (ssDNA) is an important intermediate in many DNA repair pathways. Here we describe protocols that permit the measurement of ssDNA that has arisen in the yeast genome in vivo, in response to telomere uncapping. Yeast strains defective in DNA damage response (DDR) genes can be used to infer the roles of the corresponding proteins in regulating ssDNA production and in responding to ssDNA. Using column based methods to purify yeast genomic DNA and quantitative amplification of single-stranded DNA (QAOS) it is possible to measure ssDNA at numerous single copy loci in the yeast genome. We describe how to measure ssDNA in synchronous cultures of cdc13-1 mutants, containing a temperature sensitive mutation in an essential telomere capping protein, and in asynchronous cultures of yku70Delta mutants also defective in telomere capping.

Modelling the Checkpoint Response to Telomere Uncapping in Budding Yeast

Journal of the Royal Society, Interface / the Royal Society. Feb, 2007  |  Pubmed ID: 17015293

One of the DNA damage-response mechanisms in budding yeast is temporary cell-cycle arrest while DNA repair takes place. The DNA damage response requires the coordinated interaction between DNA repair and checkpoint pathways. Telomeres of budding yeast are capped by the Cdc13 complex. In the temperature-sensitive cdc13-1 strain, telomeres are unprotected over a specific temperature range leading to activation of the DNA damage response and subsequently cell-cycle arrest. Inactivation of cdc13-1 results in the generation of long regions of single-stranded DNA (ssDNA) and is affected by the activity of various checkpoint proteins and nucleases. This paper describes a mathematical model of how uncapped telomeres in budding yeast initiate the checkpoint pathway leading to cell-cycle arrest. The model was encoded in the Systems Biology Markup Language (SBML) and simulated using the stochastic simulation system Biology of Ageing e-Science Integration and Simulation (BASIS). Each simulation follows the time course of one mother cell keeping track of the number of cell divisions, the level of activity of each of the checkpoint proteins, the activity of nucleases and the amount of ssDNA generated. The model can be used to carry out a variety of in silico experiments in which different genes are knocked out and the results of simulation are compared to experimental data. Possible extensions to the model are also discussed.

MTAS: Mental Health of Applicants Seems to Be Deteriorating

BMJ (Clinical Research Ed.). Jun, 2007  |  Pubmed ID: 17599984

Mrc1 Protects Uncapped Budding Yeast Telomeres from Exonuclease EXO1

DNA Repair. Nov, 2007  |  Pubmed ID: 17618841

Mrc1 (Mediator of Replication Checkpoint 1) is a component of the DNA replication fork machinery and is necessary for checkpoint activation after replication stress. In this study, we addressed the role of Mrc1 at uncapped telomeres. Our experiments show that Mrc1 contributes to the vitality of both cdc13-1 and yku70Delta telomere capping mutants. Cells with telomere capping defects containing MRC1 or mrc1(AQ), a checkpoint defective allele, exhibit similar growth, suggesting growth defects of cdc13-1 mrc1Delta are not due to checkpoint defects. This is in accordance with Mrc1-independent Rad53 activation after telomere uncapping. Poor growth of cdc13-1 mutants in the absence of Mrc1 is a result of enhanced single stranded DNA accumulation at uncapped telomeres. Consistent with this, deletion of EXO1, encoding a nuclease that contributes to single stranded DNA accumulation after telomere uncapping, improves growth of cdc13-1 mrc1Delta strains and decreases ssDNA production. Our observations show that Mrc1, a core component of the replication fork, plays an important role in telomere capping, protecting from nucleases and checkpoint pathways.

Histone Methyltransferase Dot1 and Rad9 Inhibit Single-stranded DNA Accumulation at DSBs and Uncapped Telomeres

The EMBO Journal. May, 2008  |  Pubmed ID: 18418382

Cells respond to DNA double-strand breaks (DSBs) and uncapped telomeres by recruiting checkpoint and repair factors to the site of lesions. Single-stranded DNA (ssDNA) is an important intermediate in the repair of DSBs and is produced also at uncapped telomeres. Here, we provide evidence that binding of the checkpoint protein Rad9, through its Tudor domain, to methylated histone H3-K79 inhibits resection at DSBs and uncapped telomeres. Loss of DOT1 or mutations in RAD9 influence a Rad50-dependent nuclease, leading to more rapid accumulation of ssDNA, and faster activation of the critical checkpoint kinase, Mec1. Moreover, deletion of RAD9 or DOT1 partially bypasses the requirement for CDK1 in DSB resection. Interestingly, Dot1 contributes to checkpoint activation in response to low levels of telomere uncapping but is not essential with high levels of uncapping. We suggest that both Rad9 and histone H3 methylation allow transmission of the damage signal to checkpoint kinases, and keep resection of damaged DNA under control influencing, both positively and negatively, checkpoint cascades and contributing to a tightly controlled response to DNA damage.

Checkpoint-dependent Phosphorylation of Exo1 Modulates the DNA Damage Response

The EMBO Journal. Sep, 2008  |  Pubmed ID: 18756267

Exo1 is a nuclease involved in mismatch repair, DSB repair, stalled replication fork processing and in the DNA damage response triggered by dysfunctional telomeres. In budding yeast and mice, Exo1 creates single-stranded DNA (ssDNA) at uncapped telomeres. This ssDNA accumulation activates the checkpoint response resulting in cell cycle arrest. Here, we demonstrate that Exo1 is phosphorylated when telomeres are uncapped in cdc13-1 and yku70Delta yeast cells, and in response to the induction of DNA damage. After telomere uncapping, Exo1 phosphorylation depends on components of the checkpoint machinery such as Rad24, Rad17, Rad9, Rad53 and Mec1, but is largely independent of Chk1, Tel1 and Dun1. Serines S372, S567, S587 and S692 of Exo1 were identified as targets for phosphorylation. Furthermore, mutation of these Exo1 residues altered the DNA damage response to uncapped telomeres and camptothecin treatment, in a manner that suggests Exo1 phosphorylation inhibits its activity. We propose that Rad53-dependent Exo1 phosphorylation is involved in a negative feedback loop to limit ssDNA accumulation and DNA damage checkpoint activation.

A Genome Wide Analysis of the Response to Uncapped Telomeres in Budding Yeast Reveals a Novel Role for the NAD+ Biosynthetic Gene BNA2 in Chromosome End Protection

Genome Biology. 2008  |  Pubmed ID: 18828915

Telomeres prevent the ends of eukaryotic chromosomes from being recognized as damaged DNA and protect against cancer and ageing. When telomere structure is perturbed, a co-ordinated series of events promote arrest of the cell cycle so that cells carrying damaged telomeres do not divide. In order to better understand the eukaryotic response to telomere damage, budding yeast strains harboring a temperature sensitive allele of an essential telomere capping gene (cdc13-1) were subjected to a transcriptomic study.

A Genomewide Suppressor and Enhancer Analysis of Cdc13-1 Reveals Varied Cellular Processes Influencing Telomere Capping in Saccharomyces Cerevisiae

Genetics. Dec, 2008  |  Pubmed ID: 18845848

In Saccharomyces cerevisiae, Cdc13 binds telomeric DNA to recruit telomerase and to "cap" chromosome ends. In temperature-sensitive cdc13-1 mutants telomeric DNA is degraded and cell-cycle progression is inhibited. To identify novel proteins and pathways that cap telomeres, or that respond to uncapped telomeres, we combined cdc13-1 with the yeast gene deletion collection and used high-throughput spot-test assays to measure growth. We identified 369 gene deletions, in eight different phenotypic classes, that reproducibly demonstrated subtle genetic interactions with the cdc13-1 mutation. As expected, we identified DNA damage checkpoint, nonsense-mediated decay and telomerase components in our screen. However, we also identified genes affecting casein kinase II activity, cell polarity, mRNA degradation, mitochondrial function, phosphate transport, iron transport, protein degradation, and other functions. We also identified a number of genes of previously unknown function that we term RTC, for restriction of telomere capping, or MTC, for maintenance of telomere capping. It seems likely that many of the newly identified pathways/processes that affect growth of budding yeast cdc13-1 mutants will play evolutionarily conserved roles at telomeres. The high-throughput spot-testing approach that we describe is generally applicable and could aid in understanding other aspects of eukaryotic cell biology.

Psychological Impact of Systemic Training Failure on Mental Health and Career Satisfaction of UK Trainees: Lessons from an Online Attitudes Survey

The International Journal of Social Psychiatry. Mar, 2009  |  Pubmed ID: 19240207

This study aims to evaluate the psychological and career-planning impact of the new postgraduate training system Modernising Medical Careers (MMC) on junior doctor applicants in the UK. We hypothesized that certain junior doctor groups were more vulnerable to distress during the process than others.

Telomere Maintenance and Survival in Saccharomyces Cerevisiae in the Absence of Telomerase and RAD52

Genetics. Jul, 2009  |  Pubmed ID: 19380905

Telomeres are essential features of linear genomes that are crucial for chromosome stability. Telomeric DNA is usually replenished by telomerase. Deletion of genes encoding telomerase components leads to telomere attrition with each cycle of DNA replication, eventually causing cell senescence or death. In the Saccharomyces cerevisiae strain W303, telomerase-null populations bypass senescence and, unless EXO1 is also deleted, this survival is RAD52 dependent. Unexpectedly, we found that the S. cerevisiae strain S288C could survive the removal of RAD52 and telomerase at a low frequency without additional gene deletions. These RAD52-independent survivors were propagated stably and exhibited a telomere organization typical of recombination between telomeric DNA tracts, and in diploids behaved as a multigenic trait. The polymerase-delta subunit Pol32 was dispensable for the maintenance of RAD52-independent survivors. The incidence of this rare escape was not affected by deletion of other genes necessary for RAD52-dependent survival, but correlated with initial telomere length. If W303 strains lacking telomerase and RAD52 first underwent telomere elongation, rare colonies could then bypass senescence. We suggest that longer telomeres provide a more proficient substrate for a novel telomere maintenance mechanism that does not rely on telomerase, RAD52, or POL32.

Taming the Tiger by the Tail: Modulation of DNA Damage Responses by Telomeres

The EMBO Journal. Aug, 2009  |  Pubmed ID: 19629039

Telomeres are by definition stable and inert chromosome ends, whereas internal chromosome breaks are potent stimulators of the DNA damage response (DDR). Telomeres do not, as might be expected, exclude DDR proteins from chromosome ends but instead engage with many DDR proteins. However, the most powerful DDRs, those that might induce chromosome fusion or cell-cycle arrest, are inhibited at telomeres. In budding yeast, many DDR proteins that accumulate most rapidly at double strand breaks (DSBs), have important functions in physiological telomere maintenance, whereas DDR proteins that arrive later tend to have less important functions. Considerable diversity in telomere structure has evolved in different organisms and, perhaps reflecting this diversity, different DDR proteins seem to have distinct roles in telomere physiology in different organisms. Drawing principally on studies in simple model organisms such as budding yeast, in which many fundamental aspects of the DDR and telomere biology have been established; current views on how telomeres harness aspects of DDR pathways to maintain telomere stability and permit cell-cycle division are discussed.

Support of Association Between BRD1 and Both Schizophrenia and Bipolar Affective Disorder

American Journal of Medical Genetics. Part B, Neuropsychiatric Genetics : the Official Publication of the International Society of Psychiatric Genetics. Mar, 2010  |  Pubmed ID: 19693800

A recent study published by our group implicated the bromodomain containing protein 1 (BRD1) gene located at chromosome 22q13.33 with schizophrenia (SZ) and bipolar affective disorder (BPD) susceptibility and provided evidence suggesting a possible role for BRD1 in neurodevelopment. The present study reports an association analysis of BRD1 and the neighboring gene ZBED4 using a Caucasian case-control sample from Denmark and England (UK/DK sample: 490 patients with BPD, 527 patients with SZ, and 601 control individuals), and genotypes obtained from a BPD genome wide association (GWA) study of an overlapping English sample comprising 506 patients with BPD and 510 control individuals (UCL sample). In the UK/DK sample we genotyped 11 SNPs in the BRD1 region, of which six showed association with SZ (minimal single marker P-values of 0.0014), including two SNPs that previously showed association in a Scottish population [Severinsen et al. (2006); Mol Psychiatry 11(12): 1126-1138]. Haplotype analysis revealed specific risk as well as protective haplotypes with a minimal P-value of 0.0027. None of the 11 SNPs showed association with BPD. However, analyzing seven BRD1 SNPs obtained from the BPD GWA study, positive associations with BPD was observed with all markers (minimal P-value of 0.0014). The associations reported add further support for the implication of BRD1 with SZ and BPD susceptibility.

Analysis of Licking Microstructure Provides No Evidence for a Reduction in Reward Value Following Acute or Sub-chronic Phencyclidine Administration

Psychopharmacology. Apr, 2010  |  Pubmed ID: 20145910

The N-methyl D-aspartate antagonist phencyclidine (PCP) is purported to mimic the negative, cognitive and positive symptoms of schizophrenia. Thus, acute and sub-chronic PCP treatment in rodents might produce anhedonia, a decrease in the pleasure produced by rewards.

Colonyzer: Automated Quantification of Micro-organism Growth Characteristics on Solid Agar

BMC Bioinformatics. 2010  |  Pubmed ID: 20509870

High-throughput screens comparing growth rates of arrays of distinct micro-organism cultures on solid agar are useful, rapid methods of quantifying genetic interactions. Growth rate is an informative phenotype which can be estimated by measuring cell densities at one or more times after inoculation. Precise estimates can be made by inoculating cultures onto agar and capturing cell density frequently by plate-scanning or photography, especially throughout the exponential growth phase, and summarising growth with a simple dynamic model (e.g. the logistic growth model). In order to parametrize such a model, a robust image analysis tool capable of capturing a wide range of cell densities from plate photographs is required.

Telomere Replication: Mre11 Leads the Way

Molecular Cell. Jun, 2010  |  Pubmed ID: 20620949

In this issue of Molecular Cell, Faure et al. (2010) establish a critical role for the Mre11 complex in the recruitment of telomerase to leading- but not lagging-strand telomeres of budding yeast.

Survival and Growth of Yeast Without Telomere Capping by Cdc13 in the Absence of Sgs1, Exo1, and Rad9

PLoS Genetics. Aug, 2010  |  Pubmed ID: 20808892

Maintenance of telomere capping is absolutely essential to the survival of eukaryotic cells. Telomere capping proteins, such as Cdc13 and POT1, are essential for the viability of budding yeast and mammalian cells, respectively. Here we identify, for the first time, three genetic modifications that allow budding yeast cells to survive without telomere capping by Cdc13. We found that simultaneous inactivation of Sgs1, Exo1, and Rad9, three DNA damage response (DDR) proteins, is sufficient to allow cell division in the absence of Cdc13. Quantitative amplification of ssDNA (QAOS) was used to show that the RecQ helicase Sgs1 plays an important role in the resection of uncapped telomeres, especially in the absence of checkpoint protein Rad9. Strikingly, simultaneous deletion of SGS1 and the nuclease EXO1, further reduces resection at uncapped telomeres and together with deletion of RAD9 permits cell survival without CDC13. Pulsed-field gel electrophoresis studies show that cdc13-1 rad9Delta sgs1Delta exo1Delta strains can maintain linear chromosomes despite the absence of telomere capping by Cdc13. However, with continued passage, the telomeres of such strains eventually become short and are maintained by recombination-based mechanisms. Remarkably, cdc13Delta rad9Delta sgs1Delta exo1Delta strains, lacking any Cdc13 gene product, are viable and can grow indefinitely. Our work has uncovered a critical role for RecQ helicases in limiting the division of cells with uncapped telomeres, and this may provide one explanation for increased tumorigenesis in human diseases associated with mutations of RecQ helicases. Our results reveal the plasticity of the telomere cap and indicate that the essential role of telomere capping is to counteract specific aspects of the DDR.

Pif1- and Exo1-dependent Nucleases Coordinate Checkpoint Activation Following Telomere Uncapping

The EMBO Journal. Dec, 2010  |  Pubmed ID: 21045806

Essential telomere 'capping' proteins act as a safeguard against ageing and cancer by inhibiting the DNA damage response (DDR) and regulating telomerase recruitment, thus distinguishing telomeres from double-strand breaks (DSBs). Uncapped telomeres and unrepaired DSBs can both stimulate a potent DDR, leading to cell cycle arrest and cell death. Using the cdc13-1 mutation to conditionally 'uncap' telomeres in budding yeast, we show that the telomere capping protein Cdc13 protects telomeres from the activity of the helicase Pif1 and the exonuclease Exo1. Our data support a two-stage model for the DDR at uncapped telomeres; Pif1 and Exo1 resect telomeric DNA <5 kb from the chromosome end, stimulating weak checkpoint activation; resection is extended >5 kb by Exo1 and full checkpoint activation occurs. Cdc13 is also crucial for telomerase recruitment. However, cells lacking Cdc13, Pif1 and Exo1, do not senesce and maintain their telomeres in a manner dependent upon telomerase, Ku and homologous recombination. Thus, attenuation of the DDR at uncapped telomeres can circumvent the need for otherwise-essential telomere capping proteins.

Analysis of Genetic Deletions and Duplications in the University College London Bipolar Disorder Case Control Sample

European Journal of Human Genetics : EJHG. May, 2011  |  Pubmed ID: 21206513

Genetic deletions and duplications known as copy number variants have been strongly implicated in genetic susceptibility to schizophrenia, autism, attention deficit hyperactivity disorder and epilepsy. The overall rate of copy number variants in the University College London (UCL) bipolar disorder sample was found to be slightly lower than the rate in controls. This finding confirms the results from other studies that have also shown no increased rate of copy number variants in bipolar disorder. However, some rare duplications and deletions were observed only in bipolar disorder cases and not in controls, these included some that had previously been detected only in rare cases of bipolar disorder. We conclude that copy-number variant analysis shows no obvious sharing of the same genetic susceptibility between schizophrenia and bipolar disorder. Copy number variants do not seem to have an important role in susceptibility to bipolar disorder, they may, however, still represent a rare cause of the disease, although the evidence for this is far from clear.

Simultaneous Contrast: Evidence from Licking Microstructure and Cross-solution Comparisons

Journal of Experimental Psychology. Animal Behavior Processes. Apr, 2011  |  Pubmed ID: 21381860

The microstructure of rats' licking responses was analyzed to investigate both "classic" simultaneous contrast (e.g., Flaherty & Largen, 1975) and a novel discrete-trial contrast procedure where access to an 8% test solution of sucrose was preceded by a sample of either 2%, 8%, or 32% sucrose (Experiments 1 and 2, respectively). Consumption of a given concentration of sucrose was higher when consumed alongside a low rather than high concentration comparison solution (positive contrast) and consumption of a given concentration of sucrose was lower when consumed alongside a high rather than a low concentration comparison solution (negative contrast). Furthermore, positive contrast increased the size of lick clusters while negative contrast decreased the size of lick clusters. Lick cluster size has a positive monotonic relationship with the concentration of palatable solutions and so positive and negative contrasts produced changes in lick cluster size that were analogous to raising or lowering the concentration of the test solution respectively. Experiment 3 utilized the discrete-trial procedure and compared contrast between two solutions of the same type (sucrose-sucrose or maltodextrin-maltodextrin) or contrast across solutions (sucrose-maltodextrin or maltodextrin-sucrose). Contrast effects on consumption were present, but reduced in size, in the cross-solution conditions. Moreover, lick cluster sizes were not affected at all by cross-solution contrasts as they were by same-solution contrasts. These results are consistent with the idea that simultaneous contrast effects depend, at least partially, on sensory mechanisms.

Lack of Allelic Association Between Markers at the DRD2 and ANKK1 Gene Loci with the Alcohol-dependence Syndrome and Criminal Activity

Psychiatric Genetics. Dec, 2011  |  Pubmed ID: 21403585

Customizable Views on Semantically Integrated Networks for Systems Biology

Bioinformatics (Oxford, England). May, 2011  |  Pubmed ID: 21414991

The rise of high-throughput technologies in the post-genomic era has led to the production of large amounts of biological data. Many of these datasets are freely available on the Internet. Making optimal use of these data is a significant challenge for bioinformaticians. Various strategies for integrating data have been proposed to address this challenge. One of the most promising approaches is the development of semantically rich integrated datasets. Although well suited to computational manipulation, such integrated datasets are typically too large and complex for easy visualization and interactive exploration.

Quantitative Fitness Analysis Shows That NMD Proteins and Many Other Protein Complexes Suppress or Enhance Distinct Telomere Cap Defects

PLoS Genetics. Apr, 2011  |  Pubmed ID: 21490951

To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70Δ). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time. The data are fitted to a logistic population growth model; and growth parameters, such as maximum growth rate and maximum doubling potential, are deduced. QFA reveals that as many as 5% of systematic gene deletions, affecting numerous functional classes, strongly interact with telomere capping defects. We show that, while Cdc13 and Yku70 perform complementary roles in telomere capping, their genetic interaction profiles differ significantly. At least 19 different classes of functionally or physically related proteins can be identified as interacting with cdc13-1, yku70Δ, or both. Each specific genetic interaction informs the roles of individual gene products in telomere biology. One striking example is with genes of the nonsense-mediated RNA decay (NMD) pathway which, when disabled, suppress the conditional cdc13-1 mutation but enhance the null yku70Δ mutation. We show that the suppressing/enhancing role of the NMD pathway at uncapped telomeres is mediated through the levels of Stn1, an essential telomere capping protein, which interacts with Cdc13 and recruitment of telomerase to telomeres. We show that increased Stn1 levels affect growth of cells with telomere capping defects due to cdc13-1 and yku70Δ. QFA is a sensitive, high-throughput method that will also be useful to understand other aspects of microbial cell biology.

Genetic Association Study of GABRA2 Single Nucleotide Polymorphisms and Electroencephalography in Alcohol Dependence

Neuroscience Letters. Aug, 2011  |  Pubmed ID: 21683760

The gamma aminobutyric acid (GABA) system has been implicated in the susceptibility to develop alcohol dependence and in determining electroencephalogram (EEG) beta activity. The role of the GABA receptor alpha-2 gene (GABRA2) in human alcohol dependence was determined in a genetic and electrophysiological study. The study population comprised 586 white UK individuals with alcohol dependence but a very low prevalence of co-morbid drug dependence, and 603 ancestrally matched healthy controls. Genotyping for seven GABRA2 single nucleotide polymorphisms (SNPs), identified from the literature as positively associated with alcohol dependence, was performed with success rates of 90% or greater. EEGs were available in 32 selected patients who had been abstinent from alcohol for a minimum of 24 months and in 138 ancestrally matched healthy controls. None of the SNPs showed allelic or haplotypic association with alcohol dependence. All markers were in Hardy Weinberg equilibrium (HWE) in the controls. HWE for marker rs279841 in the alcohol dependent sample was p=0.0199 and combined p=0.0166. Linkage disequilibrium patterns appear to be very similar to that observed in the HapMap CEU data. A significantly higher prevalence of excess EEG fast activity was found in the patients (31 vs. 14%, p=0.018). A significant relationship was found between the presence of excess EEG fast activity and GABRA2 SNPs rs548583, rs279871 and rs279841. This allelic association study provides no evidence for an association between GABRA2 polymorphisms and alcohol dependence. However, a significant relationship was identified between GABRA2 and excess EEG fast activity. This dissociation of effect may reflect the fact that the EEG is a more direct marker of phenotypic GABRA2 expression than the more heterogeneous alcohol dependence phenotype.

Confirmation of Prior Evidence of Genetic Susceptibility to Alcoholism in a Genome-wide Association Study of Comorbid Alcoholism and Bipolar Disorder

Psychiatric Genetics. Dec, 2011  |  Pubmed ID: 21876473

Alcoholism and affective disorders are both strongly comorbid and heritable. We have investigated the genetic comorbidity between bipolar affective disorder and alcoholism.

Genome-wide Analysis to Identify Pathways Affecting Telomere-initiated Senescence in Budding Yeast

G3 (Bethesda, Md.). Aug, 2011  |  Pubmed ID: 22384331

In telomerase-deficient yeast cells, like equivalent mammalian cells, telomeres shorten over many generations until a period of senescence/crisis is reached. After this, a small fraction of cells can escape senescence, principally using recombination-dependent mechanisms. To investigate the pathways that affect entry into and recovery from telomere-driven senescence, we combined a gene deletion disrupting telomerase (est1Δ) with the systematic yeast deletion collection and measured senescence characteristics in high-throughput assays. As expected, the vast majority of gene deletions showed no strong effects on entry into/exit from senescence. However, around 200 gene deletions behaving similarly to a rad52Δest1Δ archetype (rad52Δ affects homologous recombination) accelerated entry into senescence, and such cells often could not recover growth. A smaller number of strains similar to a rif1Δest1Δ archetype (rif1Δ affects proteins that bind telomeres) accelerated entry into senescence but also accelerated recovery from senescence. Our genome-wide analysis identifies genes that affect entry into and/or exit from telomere-initiated senescence and will be of interest to those studying telomere biology, replicative senescence, cancer, and ageing. Our dataset is complementary to other high-throughput studies relevant to telomere biology, genetic stability, and DNA damage responses.

Choice of Antipsychotic Treatment by European Psychiatry Trainees: Are Decisions Based on Evidence?

BMC Psychiatry. 2012  |  Pubmed ID: 22463055

Little is known about the factors influencing treatment choice in psychosis, the majority of this work being conducted with specialists (consultant) in psychiatry. We sought to examine trainees' choices of treatment for psychosis if they had to prescribe it for themselves, their patients, and factors influencing decision-making.

Similarities and Differences Between "uncapped" Telomeres and DNA Double-strand Breaks

Chromosoma. Apr, 2012  |  Pubmed ID: 22203190

Telomeric DNA is present at the ends of eukaryotic chromosomes and is bound by telomere "capping" proteins, which are the (Cdc13-Stn1-Ten1) CST complex, Ku (Yku70-Yku80), and Rap1-Rif1-Rif2 in budding yeast. Inactivation of any of these complexes causes telomere "uncapping," stimulating a DNA damage response (DDR) that frequently involves resection of telomeric DNA and stimulates cell cycle arrest. This is presumed to occur because telomeres resemble one half of a DNA double-strand break (DSB). In this review, we outline the DDR that occurs at DSBs and compare it to the DDR occurring at uncapped telomeres, in both budding yeast and metazoans. We give particular attention to the resection of DSBs in budding yeast by Mre11-Xrs2-Rad50 (MRX), Sgs1/Dna2, and Exo1 and compare their roles at DSBs and uncapped telomeres. We also discuss how resection uncapped telomeres in budding yeast is promoted by the by 9-1-1 complex (Rad17-Mec3-Ddc1), to illustrate how analysis of uncapped telomeres can serve as a model for the DDR elsewhere in the genome. Finally, we discuss the role of the helicase Pif1 and its requirement for resection of uncapped telomeres, but not DSBs. Pif1 has roles in DNA replication and mammalian and plant CST complexes have been identified and have roles in global genome replication. Based on these observations, we suggest that while the DDR at uncapped telomeres is partially due to their resemblance to a DSB, it may also be partially due to defective DNA replication. Specifically, we propose that the budding yeast CST complex has dual roles to inhibit a DSB-like DDR initiated by Exo1 and a replication-associated DDR initiated by Pif1. If true, this would suggest that the mammalian CST complex inhibits a Pif1-dependent DDR.