1,721,035 research outputs found
Repair of hydrolytic DNA deamination damage in thermophilic bacteria: cloning and characterization of a Vsr endonuclease homolog from Bacillus stearothermophilus.
No full textHydrolytic deamination of 5-methyl cytosine in double stranded DNA results in formation of a T/G mismatch that—if left unrepaired—leads to a C->T transition mutation in half of the progeny. In addition to several mismatch-specific glycosylases that have been found in both pro- and eukaryotes to channel this lesion into base excision repair by removing the T from the mismatch, Vsr endonuclease from Escherichia coli has been described which initiates repair by an endonucleolytic strand incision 5' to the mismatched T. We have isolated a gene coding for a homolog of E.coli Vsr endonuclease from the thermophilic bacterium Bacillus stearothermophilus H3 (Vsr.Bst) using a method that allows PCR amplification with degenerated primers of gene segments which code for only one highly conserved amino acid region. Vsr.Bst was produced heterologously in E.coli and purified to apparent homogeneity. Vsr.Bst specifically incises heteroduplex DNA with a preference for T/G mismatches. The selectivity of Vsr.Bst for the sequence context of the T/G mismatch appears less pronounced than for Vsr.Eco
Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination
No full textEukaryotes possess mechanisms to limit crossing over during mitotic homologous recombination, thus avoiding possible chromosomal rearrangements. We show here that budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases-Srs2 and Sgs1-that also attenuate crossing over. By chromatin immunoprecipitation, we find targeting of Mph1 to double-strand breaks in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from our analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks
Two amino acid replacements change the substrate preference of DNA mismatch glycosylase Mig.MthI from T/G to A/G.
No full textMig.MthI from Methanobacterium thermoautotrophicum and MutY of Escherichia coli are both DNA mismatch glycosylases of the ‘helix-hairpin-helix’ (HhH) superfamily of DNA repair glycosylases; the former excises thymine from T/G, the latter adenine from A/G mismatches. The structure of MutY, in complex with its low molecular weight product, adenine, has previously been determined by X-ray crystallography. Surprisingly, the set of amino acid residues of MutY that are crucial for adenine recognition is largely conserved in Mig.MthI. Here we show that replacing two amino acid residues in the (modeled) thymine binding site of Mig.MthI (Leu187 to Gln and Ala50 to Val) changes substrate discrimination between T/G and A/G by a factor of 117 in favor of the latter (from 56-fold slower to 2.1-fold faster). The Ala to Val exchange also affects T/G versus U/G selectivity. The data allow a plausible model of thymine binding and of catalytic mechanism of Mig.MthI to be constructed, the key feature of which is a bidentate hydrogen bridge of a protonated glutamate end group (number 42) with thymine centers NH-3 and O-4, with proton transfer to the exocyclic oxygen atom neutralizing the negative charge that builds up in the pyrimidine ring system as the glycosidic bond is broken in a heterolytic fashion. The results also offer an explanation for why so many different substrate specificities are realized within the HhH superfamily of DNA repair glycosylases, and they widen the scope of these enzymes as practical tools
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Helix-hairpin-helix protein MJ1434 from Methanocaldococcus jannaschii and EndoIV homologue TTC0482 from Thermus thermophilus HB27 do not process DNA uracil residues
No full textThe mutagenic threat of hydrolytic DNA cytosine deamination is met mostly by uracil DNA glycosylases (UDG) initiating base excision repair. However, several sequenced genomes of archaeal organisms are devoid of genes coding for homologues of the otherwise ubiquitous UDG superfamily of proteins. Previously, two possible solutions to this problem were offered by (i) a report of a newly discovered family of uracil DNA glycosylases exemplified by MJ1434, a protein found in the hyperthermophilic archaeon Methanocaldococcus jannaschii, and (ii) the description of TTC0482, an EndoIV homologue from the hyperthermophilic bacterium Thermus thermophilus HB27, as being able to excise uracil from DNA. Sequence homologues of both proteins can be found throughout the archaeal domain of life. Three proteins orthologous to MJ1434 and the family founder itself were tested for but failed to exhibit DNA uracil glycosylase activity when produced in an Ung-deficient Escherichia coli host. Likewise, no DNA uracil processing activity could be detected to be associated with TTC0482, while the protein was fully active as an AP endonuclease. We propose that the uracil processing activities formerly found were due to contaminations with Ung enzyme. Use of delta ung-strains as hosts for production of putatively DNA-U processing enzymes provides a simple safeguard.Georg-August-Universitat Gottinge
Towards the role of Mph1 from Saccharomyces cerevisiae and its human orthologue FANCM in the cellular genome stability network
No full textLeben ist lebensgefährlich. Die vielfältigen exogenen und endogenen Angriffe auf ihr Erbgut kann eine Zelle nur langfristig überleben, weil sie mit einem Netzwerk aufeinander abgestimmter Sicherheitsmaßnahmen ausgestattet ist: der DNA damage response. Reparaturenzyme reparieren fortlaufend DNA-Schäden, trotzdem behindern immer wieder Schäden die Replikation. Bei Replikationsproblemen helfen die Faktoren der Schadenstoleranzsysteme, indem sie die Replikation am Laufen halten und die Chromosomen in einen „vererbbaren“ Zustand bringen. Die Koordination übernimmt der DNA damage checkpoint. Ein kleines Rad im Gefüge der DNA damage response bilden die Mitglieder der FANCM-Familie, die als Motorproteine DNA-Substrate umstrukturieren können. In der Hefe Saccharomyces cerevisiae ist dies Mph1, im Menschen die weitaus größere und multifunktionalere Fanconi-Anämie-Determinante M (FANCM), die zwar gar keine Fanconi-Anämie (FA) determiniert, aber ein wichtiger Tumorsuppressor ist. FANCM orchestriert zusammen mit den anderen FA-Proteinen die Reparatur von interstrand crosslinks. Dies ist aber bei weitem nicht die einzige Funktion, die FANCM zugeschrieben wird: komplexe Aufgaben innerhalb und außerhalb des FA-Wegs sind bekannt. Die Vielfältigkeit dieser Funktionen führte zu der Hypothese, dass das Produkt der Transkriptvariante FANCM-669 möglicherweise zum breiten Spektrum der FANCM-Aktivitäten beiträgt. Meine Daten konnten dies leider nicht bestätigen. Stattdessen habe ich andere spontane Transkriptvarianten von FANCM nachweisen können (FANCM-Ex11b, FANCM-Δ3, FANCM-Δ5 und FANCM-Δ21/22), die teilweise krankheitsauslösenden, trunkierenden Genvarianten ähneln, allerdings noch einer genaueren Untersuchung bedürfen. Das Bäckerhefe-Ortholog Mph1 ist kleiner als FANCM, dafür aber eine funktionale Helikase. Als solche übernimmt Mph1 in der Zelle vielfältige Funktionen, die vor allem durch die Auflösung spezieller Rekombinationsintermediate (D-loops) bedingt sind. Bei der fehlerfreien Umgehung replikations-arretierender Schäden ist Mph1 in dieser Funktion ebenfalls unterwegs. Da es aufgrund seines namensgebenden Mutatorphänotyps noch weitere Funktionen ausüben muss, wurde im Rahmen dieser Arbeit eine mögliche Regulation von Mph1 durch Phosphorylierung untersucht. Meine Daten lieferten allerdings keinen Hinweis auf eine entscheidende Regulation durch Phosphorylierung von Mph1 im Kontext der Schadenstoleranz. Im Zuge meiner Experimente habe ich aber ein methodisches Problem bei der Messung von Schwesterchromatid-Interaktionen aufgrund multipler Integration des Reporter-konstrukts aufgedeckt, woraufhin die zu Mph1 vorliegenden Daten teilweise überprüft werden mussten. Demnach reduziert Mph1 die durch DNA-Schäden induzierten Schwesterchromatid-Interaktionen weniger stark als bisher angenommen, was aber seine postulierte Funktion nicht grundsätzlich in Frage stellt. Die Möglichkeit der multiplen Integration bestimmter Vektoren sollte aber stets bedacht werden, wenn die integrierte DNA für einen quantitativen readout verwendet wird. Zusätzlich lieferte die Restriktion der Expression von MPH1 in die G2/M-Phase Hinweise darauf, dass Mph1 in der S-Phase entbehrlich ist. Das stellt allerdings in Frage, inwiefern die für Mph1 im Kontext verschiedener Reparatur- und Schadenstoleranzwege postulierte Funktion zum Schutz arretierter Replikationsgabeln während der S Phase für das Zellüberleben relevant ist.Living is life-threatening. Cells can only endure the diverse exogenous and endogenous attacks on their genome because they are equipped with a network of coordinated security measures: the DNA damage response. Repair enzymes constantly fix the genetic material, yet persisting damages regularly impede DNA replication. Damage tolerance factors help with these replication problems by keeping replication running and putting the chromosomes into a “hereditable” state. Fine-tuned coordination is achieved by the DNA damage checkpoint. The members of the FANCM family, which as motor proteins can restructure DNA substrates, represent one cog in the framework of the DNA damage response: in the yeast Saccharomyces cerevisiae this is Mph1, in humans the larger and multifunctional Fanconi anemia determinant M (FANCM), which does not actually determine Fanconi anemia (FA) but is an ever-more-relevant tumor suppressor. FANCM, together with the other FA proteins, orchestrates the repair of interstrand crosslinks. But this is far from being the only function attributed to FANCM: complex tasks inside and outside the FA pathway are described. The diversity of these functions led to the hypothesis that the product of the transcript variant FANCM-669 might contribute to the broad spectrum of FANCM activities. Unfortunately, my data could not confirm this. Instead, I was able to detect other spontaneous transcript variants of FANCM (FANCM-Ex11b, FANCM-Δ3, FANCM-Δ5 and FANCM-Δ21/22), some of which resemble disease-causing, truncating gene variants, but still require more detailed investigation. The baker's yeast ortholog Mph1 is smaller than FANCM but a functional helicase. As such, Mph1 takes on diverse functions, which are primarily due to the dissolution of special recombination intermediates (D-loops), one example being its role in error-free bypass of replication-arresting damage. Since it has to perform more intricate functions due to its eponymous mutator phenotype, a possible regulation of Mph1 by phosphorylation was investigated here. Taken together, my data did not provide any evidence for a crucial regulation by phosphorylation of Mph1 in the context of damage tolerance. Over the course of my experiments, however, I uncovered a methodological problem in measuring sister chromatid interactions due to multiple integration of the reporter construct. Thereupon, the data available for Mph1 had to be partially reassessed. According to this, Mph1 reduces sister chromatid interactions induced by DNA damage less than previously assumed, which does not fundamentally question its postulated function though. Still, the possibility of multiple integration of certain vectors should always be considered if the integrated DNA is used for a quantitative readout. Additionally, the restriction of MPH1 expression to G2/M phase provided evidence that Mph1 is dispensable in S phase. This questions to which extent the protection of arrested replication forks during S phase postulated for Mph1 in the context of various repair and damage tolerance pathways is relevant for cell survival
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
- …
