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Subject = Saccharomyces-cerevisiae;
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Displaying Results 1 - 25 of 33 on page 1 of 2
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53bp1: function and mechanisms of focal recruitment
(2018)
FitzGerald, Jennifer E.; Grenon, Muriel; Lowndes, Noel F.
53bp1: function and mechanisms of focal recruitment
(2018)
FitzGerald, Jennifer E.; Grenon, Muriel; Lowndes, Noel F.
Abstract:
53BP1 (p53-binding protein 1) is classified as a mediator/adaptor of the DNA-damage response, and is recruited to nuclear structures termed foci following genotoxic insult. In the present paper, we review the functions of S3BP1 in DNA-damage checkpoint activation and DNA repair, and the mechanisms of its recruitment and activation following DNA damage. We focus in particular on the role of covalent histone modifications in this process.
http://hdl.handle.net/10379/11485
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Analysis of scc1-deficient cells defines a key metaphase role of vertebrate cohesin in linking sister kinetochores
(2018)
Vagnarelli, Paola; Morrison, Ciaran; Dodson, Helen; Sonoda, Eiichiro; Takeda, Shunichi;...
Analysis of scc1-deficient cells defines a key metaphase role of vertebrate cohesin in linking sister kinetochores
(2018)
Vagnarelli, Paola; Morrison, Ciaran; Dodson, Helen; Sonoda, Eiichiro; Takeda, Shunichi; Earnshaw, William C
Abstract:
Cleavage of the cohesin subunit Scc1p/Mcd1p/Rad21 permits sister chromatid separation and is considered to trigger anaphase onset. It has also been suggested that the cohesin complex is essential for chromosome condensation and for assembling fully functional kinetochores. Here, we used vertebrate cells conditionally deficient in Scc1 to probe cohesin function in mitosis. Cells lacking cohesin arrest in prometaphase, with many chromosomes failing to align at a metaphase plate and high levels of the spindle assembly checkpoint protein, BubR1, at all kinetochores. We show that the structural integrity of chromosomes is normal in the absence of Scc1. Furthermore, specific inhibition of topoisomerase II, which is required for decatenation of replicated chromosomes, can bypass the cohesin requirement for metaphase chromosome alignment and spindle checkpoint silencing. Since the kinetochore effects of Scc1 deficiency can be compensated for by topoisomerase II inhibition, we conclude that ...
http://hdl.handle.net/10379/9951
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Atr activates the s-m checkpoint during unperturbed growth to ensure sufficient replication prior to mitotic onset
(2018)
Eykelenboom, John Kenneth; Harte, Emma Christina; Canavan, Lynn; Pastor-Peidro, Ana; Ca...
Atr activates the s-m checkpoint during unperturbed growth to ensure sufficient replication prior to mitotic onset
(2018)
Eykelenboom, John Kenneth; Harte, Emma Christina; Canavan, Lynn; Pastor-Peidro, Ana; Calvo-Asensio, Irene; Llorens-Agost, Marta; Lowndes, Noel Francis
Abstract:
Cells must accurately replicate and segregate their DNA once per cell cycle in order to successfully transmit genetic information. During S phase in the presence of agents that cause replication stress, ATR-dependent checkpoints regulate origin firing and the replication machinery as well as prevent untimely mitosis. Here, we investigate the role of ATR during unperturbed growth in vertebrate cells. In the absence of ATR, individual replication forks progress more slowly, and an increased number of replication origins are activated. These cells also enter mitosis early and divide more rapidly, culminating in chromosome bridges and laggards at anaphase, failed cytokinesis, and cell death. Interestingly, cell death can be rescued by prolonging mitosis with partial inhibition of the mitotic cyclin-dependent kinase 1. Our data indicate that one of the essential roles of ATR during normal growth is to minimize the level of unreplicated DNA before the onset of mitosis.
http://hdl.handle.net/10379/11382
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Budding yeast rad9 is an atp-dependent rad53 activating machine
(2018)
Gilbert, Christopher S; Green, Catherine M; Lowndes, Noel F
Budding yeast rad9 is an atp-dependent rad53 activating machine
(2018)
Gilbert, Christopher S; Green, Catherine M; Lowndes, Noel F
Abstract:
We find budding yeast Rad9 in two distinct, large, and soluble complexes in cell extracts. The larger (greater than or equal to 850 kDa) complex, found in nondamaged cells, contains hypophosphorylated Rad9, whereas the smaller (560 kDa) complex, which forms after DNA damage, contains hyperphosphorylated Rad9 and Rad53. This smaller Rad9 complex is capable of catalyzing phosphorylation and release of active Rad53 kinase, a process requiring the kinase activity of Rad53. However, Mec1 and Tel1 are no longer required once the 560 kDa complex has been formed. We propose a model whereby Mec1/Tel1-dependent hyperphosphorylation of Rad9 results in formation of the smaller Rad9 complex and recruitment of Rad53. This complex then catalyzes activation of Rad53 by acting as a scaffold that brings Rad53 molecules into close proximity, facilitating Rad53 in trans autophosphorylation and subsequent release of activated Rad53.
http://hdl.handle.net/10379/9215
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Cell cycle-dependent formation of Cdc45-Claspin complexes in human cells is compromized by UV-mediated DNA damage
(2021)
Broderick, Ronan; Rainey, Michael D.; Santocanale, Corrado; Nasheuer, Heinz-Peter
Cell cycle-dependent formation of Cdc45-Claspin complexes in human cells is compromized by UV-mediated DNA damage
(2021)
Broderick, Ronan; Rainey, Michael D.; Santocanale, Corrado; Nasheuer, Heinz-Peter
Abstract:
The replication factor Cdc45 has essential functions in the initiation and elongation steps of eukaryotic DNA replication and plays an important role in the intra-S-phase checkpoint. Its interactions with other replication proteins during the cell cycle and after intra-S-phase checkpoint activation are only partially characterized. In the present study, we show that the C terminal part of Cdc45 may mediate its interactions with Claspin. The interactions of human Cdc45 with the three replication factors Claspin, replication protein A and DNA polymerase are maximal during the S phase. Following UVC-induced DNA damage, Cdc45-Claspin complex formation is reduced, whereas the binding of Cdc45 to replication protein A is not affected. We also show that treatment of cells with UCN-01 and phosphatidylinositol 3-kinase-like kinase inhibitors does not rescue the UV-induced destabilization of Cdc45-Claspin interactions, suggesting that the loss of the interaction between Cdc45 and Claspin occu...
http://hdl.handle.net/10379/16421
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Coordinated regulation of replication protein a activities by its subunits p14 and p32
(2018)
Weisshart, Klaus; Pestryakov, Pavel; Smith, Richard W. P.; Hartmann, Hella; Kremmer, El...
Coordinated regulation of replication protein a activities by its subunits p14 and p32
(2018)
Weisshart, Klaus; Pestryakov, Pavel; Smith, Richard W. P.; Hartmann, Hella; Kremmer, Elisabeth; Lavrik, Olga; Nasheuer, Heinz-Peter
Abstract:
The heterotrimeric replication protein A (RPA) has multiple essential activities in eukaryotic DNA metabolism and in signaling pathways. Despite extensive analyses, the functions of the smallest RPA subunit p14 are still unknown. To solve this issue we produced and characterized a dimeric RPA complex lacking p14, RPADeltap14, consisting of p70 and p32. RPADeltap14 was able to bind single-stranded DNA, but its binding mode and affinity differed from those of the heterotrimeric complex. Moreover, in the RPADeltap14 complex p32 only minimally recognized the 3'-end of a primer in a primer-template junction. Partial proteolytic digests revealed that p14 and p32 together stabilize the C terminus of p70 against degradation. Although RPADeltap14 efficiently supported bidirectional unwinding of double-stranded DNA and interacted with both the simian virus 40 (SV40) large T antigen and cellular DNA polymerase alpha-primase, it did not support cell-free SV40 DNA replication. This inabilit...
http://hdl.handle.net/10379/9979
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Distinctive activities of dna polymerases during human dna replication
(2018)
Rytkonen, Anna K.; Vaara, Markku; Nethanel, Tamar; Kaufmann, Gabriel; Sormunen, Raija; ...
Distinctive activities of dna polymerases during human dna replication
(2018)
Rytkonen, Anna K.; Vaara, Markku; Nethanel, Tamar; Kaufmann, Gabriel; Sormunen, Raija; Laara, Esa; Nasheuer, Heinz-Peter; Rahmeh, Amal; Lee, Marietta Y. W. T.; Syvaoja, Juhani E.; Pospiech, Helmut
Abstract:
The contributions of human DNA polymerases (pols) alpha, delta and epsilon during S-phase progression were studied in order to elaborate how these enzymes co-ordinate their functions during nuclear DNA replication. Pol delta was three to four times more intensely UV cross-linked to nascent DNA in late compared with early S phase, whereas the cross-linking of pols alpha and epsilon remained nearly constant throughout the S phase. Consistently, the chromatin-bound fraction of pol delta, unlike pols alpha and epsilon, increased in the late S phase. Moreover, pol delta neutralizing antibodies inhibited replicative DNA synthesis most efficiently in late S-phase nuclei, whereas antibodies against pol epsilon were most potent in early S phase. Ultrastructural localization of the pols by immuno-electron microscopy revealed pol epsilon to localize predominantly to ring-shaped clusters at electron-dense regions of the nucleus, whereas pol delta was mainly dispersed on fibrous structures. Pol ...
http://hdl.handle.net/10379/9837
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Dna polymerase ε associates with the elongating form of rna polymerase ii and nascent transcripts
(2018)
Rytkönen, Anna K.; Hillukkala, Tomi; Vaara, Markku; Sokka, Miiko; Jokela, Maarit; Sormu...
Dna polymerase ε associates with the elongating form of rna polymerase ii and nascent transcripts
(2018)
Rytkönen, Anna K.; Hillukkala, Tomi; Vaara, Markku; Sokka, Miiko; Jokela, Maarit; Sormunen, Raija; Nasheuer, Heinz-Peter; Nethanel, Tamar; Kaufmann, Gabriel; Pospiech, Helmut; Syväoja, Juhani E.
Abstract:
DNA polymerase epsilon co-operates with polymerases alpha and delta in the replicative DNA synthesis of eukaryotic cells. We describe here a specific physical interaction between DNA polymerase epsilon and RNA polymerase II, evidenced by reciprocal immunoprecipitation experiments. The interacting RNA polymerase II was the hyperphosphorylated IIO form implicated in transcriptional elongation, as inferred from (a) its reduced electrophoretic mobility that was lost upon phosphatase treatment, (b) correlation of the interaction with phosphorylation of Ser5 of the C-terminal domain heptapeptide repeat, and (c) the ability of C-terminal domain kinase inhibitors to abolish it. Polymerase epsilon was also shown to UV crosslink specifically alpha-amanitin-sensitive transcripts, unlike DNA polymerase alpha that crosslinked only to RNA-primed nascent DNA. Immunofluorescence microscopy revealed partial colocalization of RNA polymerase IIO and DNA polymerase epsilon, and immunoelectron microscop...
http://hdl.handle.net/10379/9836
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Evidence of efficient stop codon readthrough in four mammalian genes
(2014)
Loughran, Gary; Chou, Ming-Yuan; Ivanov, Ivaylo P.; Jungreis, Irwin; Kellis, Manolis; K...
Evidence of efficient stop codon readthrough in four mammalian genes
(2014)
Loughran, Gary; Chou, Ming-Yuan; Ivanov, Ivaylo P.; Jungreis, Irwin; Kellis, Manolis; Kiran, Anmol M.; Baranov, Pavel V.; Atkins, John F.
Abstract:
Stop codon readthrough is used extensively by viruses to expand their gene expression. Until recent discoveries in Drosophila, only a very limited number of readthrough cases in chromosomal genes had been reported. Analysis of conserved protein coding signatures that extend beyond annotated stop codons identified potential stop codon readthrough of four mammalian genes. Here we use a modified targeted bioinformatic approach to identify a further three mammalian readthrough candidates. All seven genes were tested experimentally using reporter constructs transfected into HEK-293T cells. Four displayed efficient stop codon readthrough, and these have UGA immediately followed by CUAG. Comparative genomic analysis revealed that in the four readthrough candidates containing UGA-CUAG, this motif is conserved not only in mammals but throughout vertebrates with the first six of the seven nucleotides being universally conserved. The importance of the CUAG motif was confirmed using a systemati...
http://hdl.handle.net/10468/5016
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Extracellular vesicles from paracoccidioides pathogenic species transport polysaccharide and expose ligands for dc-sign receptors
(2018)
da Silva, Roberta Peres; Heiss, Christian; Black, Ian; Azadi, Parastoo; Gerlach, Jared ...
Extracellular vesicles from paracoccidioides pathogenic species transport polysaccharide and expose ligands for dc-sign receptors
(2018)
da Silva, Roberta Peres; Heiss, Christian; Black, Ian; Azadi, Parastoo; Gerlach, Jared Q.; Travassos, Luiz R.; Joshi, Lokesh; Kilcoyne, Michelle; Puccia, Rosana
Abstract:
Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man, and Gal comprised most probably two major components: a high molecular mass 4,6-alpha-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-alpha-Manp main chain linked to beta-Galf (1,3) and alpha-Manp (1,6) end units. The results also suggested the presence of small amounts of a (1 -> 6)-Manp polymer, (1 -> 3)-glucan and (1 -> 6)-glucan. Glycan microarrays allowed ...
http://hdl.handle.net/10379/11011
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Fungal enzymes for bio-products from sustainable and waste biomass
(2018)
Gupta, Vijai K.; Kubicek, Christian P.; Berrin, Jean-Guy; Wilson, David W.; Couturier, ...
Fungal enzymes for bio-products from sustainable and waste biomass
(2018)
Gupta, Vijai K.; Kubicek, Christian P.; Berrin, Jean-Guy; Wilson, David W.; Couturier, Marie; Berlin, Alex; Filho, Edivaldo X.F.; Ezeji, Thaddeus
Abstract:
Lignocellulose, the most abundant renewable carbon source on earth, is the logical candidate to replace fossil carbon as the major biofuel raw material. Nevertheless, the technologies needed to convert lignocellulose into soluble products that can then be utilized by the chemical or fuel industries face several challenges. Enzymatic hydrolysis is of major importance, and we review the progress made in fungal enzyme technology over the past few years with major emphasis on (i) the enzymes needed for the conversion of polysaccharides (cellulose and hemicellulose) into soluble products, (ii) the potential uses of lignin degradation products, and (iii) current progress and bottlenecks for the use of the soluble lignocellulose derivatives in emerging biorefineries.
http://hdl.handle.net/10379/11775
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Heritability in the efficiency of nonsense-mediated mrna decay in humans
(2018)
Seoighe, Cathal; Gehring, Chris
Heritability in the efficiency of nonsense-mediated mrna decay in humans
(2018)
Seoighe, Cathal; Gehring, Chris
Abstract:
Background: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. Principal Findings: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of h...
http://hdl.handle.net/10379/13858
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Identification of evolutionarily conserved non-AUG-initiated N-terminal extensions in human coding sequences
(2011)
Ivanov, Ivaylo P.; Firth, Andrew E.; Michel, Audrey M.; Atkins, John F.; Baranov, Pavel V.
Identification of evolutionarily conserved non-AUG-initiated N-terminal extensions in human coding sequences
(2011)
Ivanov, Ivaylo P.; Firth, Andrew E.; Michel, Audrey M.; Atkins, John F.; Baranov, Pavel V.
Abstract:
In eukaryotes, it is generally assumed that translation initiation occurs at the AUG codon closest to the messenger RNA 5' cap. However, in certain cases, initiation can occur at codons differing from AUG by a single nucleotide, especially the codons CUG, UUG, GUG, ACG, AUA and AUU. While non-AUG initiation has been experimentally verified for a handful of human genes, the full extent to which this phenomenon is utilized-both for increased coding capacity and potentially also for novel regulatory mechanisms-remains unclear. To address this issue, and hence to improve the quality of existing coding sequence annotations, we developed a methodology based on phylogenetic analysis of predicted 5' untranslated regions from orthologous genes. We use evolutionary signatures of protein-coding sequences as an indicator of translation initiation upstream of annotated coding sequences. Our search identified novel conserved potential non-AUG-initiated N-terminal extensions in 42 human ...
http://hdl.handle.net/10468/5024
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Increased sister chromatid cohesion and dna damage response factor localization at an enzyme-induced dna double-strand break in vertebrate cells
(2018)
Dodson, Helen; Morrison, Ciaran G.
Increased sister chromatid cohesion and dna damage response factor localization at an enzyme-induced dna double-strand break in vertebrate cells
(2018)
Dodson, Helen; Morrison, Ciaran G.
Abstract:
The response to DNA damage in vertebrate cells involves successive recruitment of DNA signalling and repair factors. We used light microscopy to monitor the genetic dependencies of such localization to a single, induced DNA double strand break (DSB) in vertebrate cells. We used an inducible version of the rare-cutting I-SceI endonuclease to cut a chromosomally integrated I-SceI site beside a Tet operator array that was visualized by binding a Tet repressor-GFP fusion. Formation of gamma-H2AX foci at a single DSB was independent of ATM or Ku70. ATM-deficient cells showed normal kinetics of 53Bp1 recruitment to DSBs, but Rad51 localization was retarded. 53Bp1 and Rad51 foci formation at a single DSB was greatly reduced in H2AX-null DT40 cells. We also observed decreased inter-sister chromatid distances after DSB induction, suggesting that cohesin loading at DSBs causes elevated sister chromatid cohesion. Loss of ATM reduced DSB-induced cohesion, consistent with cohesin being an ATM ta...
http://hdl.handle.net/10379/11212
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Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose
(2014)
Ali, Shahin S.; Nugent, Brian; Mullins, Ewen; Doohan, Fiona M.
Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose
(2014)
Ali, Shahin S.; Nugent, Brian; Mullins, Ewen; Doohan, Fiona M.
Abstract:
Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for gl...
http://hdl.handle.net/10197/6129
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Interplay of dna damage and cell cycle signaling at the level of human replication protein a
(2018)
Borgstahl, Gloria E.O.; Brader, Kerry; Mosel, Adam; Liu, Shengqin; Kremmer, Elisabeth; ...
Interplay of dna damage and cell cycle signaling at the level of human replication protein a
(2018)
Borgstahl, Gloria E.O.; Brader, Kerry; Mosel, Adam; Liu, Shengqin; Kremmer, Elisabeth; Goettsch, Kaitlin A.; Kolar, Carol; Nasheuer, Heinz-Peter; Oakley, Greg G.
http://hdl.handle.net/10379/10482
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Mechanisms for atp-dependent chromatin remodelling: the means to the end
(2018)
Flaus, Andrew; Owen-Hughes, Tom
Mechanisms for atp-dependent chromatin remodelling: the means to the end
(2018)
Flaus, Andrew; Owen-Hughes, Tom
Abstract:
Chromatin remodelling is the ATP-dependent change in nucleosome organisation driven by Snf2 family ATPases. The biochemistry of this process depends on the behaviours of ATP-dependent motor proteins and their dynamic nucleosome substrates, which brings significant technical and conceptual challenges. Steady progress has been made in characterising the polypeptides of which these enzymes are comprised. Divergence in the sequences of different subfamilies of Snf2-related proteins suggests that the motors are adapted for different functions. Recently, structural insights have suggested that the Snf2 ATPase acts as a context-sensitive DNA translocase. This may have arisen as a means to enable efficient access to DNA in the high density of the eukaryotic nucleus. How the enzymes engage nucleosomes and how the network of noncovalent interactions within the nucleosome respond to the force applied remains unclear, and it remains prudent to recognise the potential for both DNA distortions an...
http://hdl.handle.net/10379/11511
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Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus talaromyces emersonii
(2018)
Maloney, Alan P.; Callan, Susan M.; Murray, Patrick G.; Tuohy, Maria G.
Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus talaromyces emersonii
(2018)
Maloney, Alan P.; Callan, Susan M.; Murray, Patrick G.; Tuohy, Maria G.
Abstract:
Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 degreesC in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The full-length m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal spe...
http://hdl.handle.net/10379/9482
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Mrc1, tof1 and csm3 inhibit cag·ctg repeat instability by at least two mechanisms
(2018)
Razidlo, David F.; Lahue, Robert S.
Mrc1, tof1 and csm3 inhibit cag·ctg repeat instability by at least two mechanisms
(2018)
Razidlo, David F.; Lahue, Robert S.
Abstract:
Trinucleotide repeats frequently expand and contract in humans and model organisms. Protein factors that modulate this process have been found by candidate gene approaches or mutant screens for increased expansion rates. To extend this effort, Saccharomyces cerevisiae mutants with higher CAG.CTG repeat contraction rates were sought using a disruption library. This screen identified Mrc1, the homolog of human Claspin, which mediates the replication and DNA damage checkpoints, and also couples the replicative helicase and polymerase. Genetic analysis showed that Mrc1, along with Tof1 and Csm3, inhibits instability in two distinct ways. Contraction rates Of (CAG)(20) tracts are elevated by loss of Mrc1, Tof1 or Csm3, but not by defects in most replication checkpoint or DNA damage checkpoint proteins. The three proteins likely inhibit contractions primarily through their coupling activity, which would prevent accumulation of single-strand template DNA prior to the formation of aberrant ...
http://hdl.handle.net/10379/13606
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Mutsβ and histone deacetylase complexes promote expansions of trinucleotide repeats in human cells
(2018)
Gannon, Anne-Marie M.; Frizzell, Aisling; Healy, Evan; Lahue, Robert S.
Mutsβ and histone deacetylase complexes promote expansions of trinucleotide repeats in human cells
(2018)
Gannon, Anne-Marie M.; Frizzell, Aisling; Healy, Evan; Lahue, Robert S.
Abstract:
Trinucleotide repeat (TNR) expansions cause at least 17 heritable neurological diseases, including Huntington's disease. Expansions are thought to arise from abnormal processing of TNR DNA by specific trans-acting proteins. For example, the DNA repair complex MutS beta (MSH2-MSH3 heterodimer) is required in mice for on-going expansions of long, disease-causing alleles. A distinctive feature of TNR expansions is a threshold effect, a narrow range of repeat units (similar to 30-40 in humans) at which mutation frequency rises dramatically and disease can initiate. The goal of this study was to identify factors that promote expansion of threshold-length CTG center dot CAG repeats in a human astrocytic cell line. siRNA knockdown of the MutS beta subunits MSH2 or MSH3 impeded expansions of threshold-length repeats, while knockdown of the MutS alpha subunit MSH6 had no effect. Chromatin immunoprecipitation experiments indicated that MutS beta, but not MutS alpha, was enriched at the T...
http://hdl.handle.net/10379/11579
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Regulation of cdc45 in the cell cycle and after dna damage
(2018)
Broderick, Ronan; Nasheuer, Heinz-Peter
Regulation of cdc45 in the cell cycle and after dna damage
(2018)
Broderick, Ronan; Nasheuer, Heinz-Peter
Abstract:
The Cdc (cell division cycle) 45 protein has a central role in the regulation of the initiation and elongation stages of eukaryotic chromosomal DNA replication. In addition, it is the main target for a Chk1 (checkpoint kinase i)-dependent Cdc25/CDK2 (cyclin-dependent kinase 2)-independent DNA damage checkpoint signal transduction pathway following low doses of 8PDE (benzo[a]pyrene dihydrodiol epoxide) treatment, which causes DNA damage similar to UV-induced adducts. cdc45 interacts physically and functionally with the putative eukaryotic replicative DNA helicase, the MCM (mini-chromosome maintenance) complex, and forms a helicase active 'supercomplex', the CMG [cdc45-MCM2-7-GINS (go-ichi-ni-san)] complex. These known protein-protein interactions, as well as unknown interactions and post-translational modifications, may be important for the regulation of Cdc45 and the initiation of DNA replication following DNA damage. Future studies will help to elucidate the molecular bas...
http://hdl.handle.net/10379/10543
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Regulation of the dna damage response and gene expression by the dot1l histone methyltransferase and the 53bp1 tumour suppressor
(2018)
FitzGerald, Jennifer; Moureau, Sylvie; Drogaris, Paul; O'Connell, Enda; Abshiru, N...
Regulation of the dna damage response and gene expression by the dot1l histone methyltransferase and the 53bp1 tumour suppressor
(2018)
FitzGerald, Jennifer; Moureau, Sylvie; Drogaris, Paul; O'Connell, Enda; Abshiru, Nebiyu; Verreault, Alain; Thibault, Pierre; Grenon, Muriel; Lowndes, Noel F.
Abstract:
Background: Dot1L, a histone methyltransferase that targets histone H3 lysine 79 (H3K79), has been implicated in gene regulation and the DNA damage response although its functions in these processes remain poorly defined. Methodology/Principal Findings: Using the chicken DT40 model system, we generated cells in which the Dot1L gene is disrupted to examine the function and focal recruitment of the 53Bp1 DNA damage response protein. Detailed kinetic and dose response assays demonstrate that, despite the absence of H3K79 methylation demonstrated by mass spectrometry, 53Bp1 focal recruitment is not compromised in these cells. We also describe, for the first time, the phenotypes of a cell line lacking both Dot1L and 53Bp1. Dot1L(-/-) and wild type cells are equally resistant to ionising radiation, whereas 53Bp1(-/-)/Dot1L(-/-) cells display a striking DNA damage resistance phenotype. Dot1L and 53Bp1 also affect the expression of many genes. Loss of Dot1L activity dramatically alters the ...
http://hdl.handle.net/10379/11483
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Repression of g1/s transcription is mediated via interaction of the gtb motifs of nrm1 and whi5 with swi6
(2018)
Travesa, A.; Kalashnikova, T. I.; de Bruin, R. A. M.; Cass, S. R.; Chahwan, C.; Lee, D....
Repression of g1/s transcription is mediated via interaction of the gtb motifs of nrm1 and whi5 with swi6
(2018)
Travesa, A.; Kalashnikova, T. I.; de Bruin, R. A. M.; Cass, S. R.; Chahwan, C.; Lee, D. E.; Lowndes, N. F.; Wittenberg, C.
Abstract:
In Saccharomyces cerevisiae, G(1)/S transcription factors MBF and SBF regulate a large family of genes important for entry to the cell cycle and DNA replication and repair. Their regulation is crucial for cell viability, and it is conserved throughout evolution. MBF and SBF consist of a common component, Swi6, and a DNA-specific binding protein, Mbp1 and Swi4, respectively. Transcriptional repressors bind to and regulate the activity of both transcription factors. Whi5 binds to SBF and represses its activity at the beginning of the G(1) phase to prevent early activation. Nrm1 binds to MBF to repress transcription as cells progress through S phase. Here, we describe a protein motif, the GTB motif (for G(1)/S transcription factor binding), in Nrm1 and Whi5 that is required to bind to the transcription factors. We also identify a region of the carboxy terminus of Swi6 that is required for Nrm1 and Whi5 binding to their target transcription factors and show that mutation of this region ...
http://hdl.handle.net/10379/14192
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Roles of vertebrate smc5 in sister chromatid cohesion and homologous recombinational repair
(2018)
Stephan, A. K.; Kliszczak, M.; Dodson, H.; Cooley, C.; Morrison, C. G.
Roles of vertebrate smc5 in sister chromatid cohesion and homologous recombinational repair
(2018)
Stephan, A. K.; Kliszczak, M.; Dodson, H.; Cooley, C.; Morrison, C. G.
Abstract:
The structural maintenance of chromosomes (Smc) family members Smc5 and Smc6 are both essential in budding and fission yeasts. Yeast smc5/6 mutants are hypersensitive to DNA damage, and Smc5/6 is recruited to HO-induced double-strand breaks (DSBs), facilitating intersister chromatid recombinational repair. To determine the role of the vertebrate Smc5/6 complex during the normal cell cycle, we generated an Smc5-deficient chicken DT40 cell line using gene targeting. Surprisingly, Smc5(-) cells were viable, although they proliferated more slowly than controls and showed mitotic abnormalities. Smc5-deficient cells were sensitive to methyl methanesulfonate and ionizing radiation (IR) and showed increased chromosome aberration levels upon irradiation. Formation and resolution of Rad51 and gamma-H2AX foci after irradiation were altered in Smc5 mutants, suggesting defects in homologous recombinational (HR) repair of DNA damage. Ku70(-/-) Smc5(-) cells were more sensitive to IR than either s...
http://hdl.handle.net/10379/14011
Marked
Mark
Segregation of replicative dna polymerases during s phase
(2018)
Vaara, Markku; Itkonen, Harri; Hillukkala, Tomi; Liu, Zhe; Nasheuer, Heinz-Peter; Schaa...
Segregation of replicative dna polymerases during s phase
(2018)
Vaara, Markku; Itkonen, Harri; Hillukkala, Tomi; Liu, Zhe; Nasheuer, Heinz-Peter; Schaarschmidt, Daniel; Pospiech, Helmut; Syväoja, Juhani E.
Abstract:
DNA polymerases (Pol) alpha, delta, and is an element of replicate the bulk of chromosomal DNA in eukaryotic cells, Pol is an element of being the main leading strand and Pol delta the lagging strand DNA polymerase. By applying chromatin immunoprecipitation (ChIP) and quantitative PCR we found that at G(1)/S arrest, all three DNA polymerases were enriched with DNA containing the early firing lamin B2 origin of replication and, 2 h after release from the block, with DNA containing the origin at the upstream promoter region of the MCM4 gene. Pol alpha, delta, and is an element of were released from these origins upon firing. All three DNA polymerases, Mcm3 and Cdc45, but not Orc2, still formed complexes in late S phase. Reciprocal ChIP of the three DNA polymerases revealed that at G(1)/S arrest and early in S phase, Pol alpha, delta, and is an element of were associated with the same nucleoprotein complexes, whereas in late S phase Pol is an element of and Pol alpha/delta were largely...
http://hdl.handle.net/10379/14247
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NUI Galway (30)
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2021 (1)
2018 (29)
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