Gemcitabine Functions Epigenetically by Inhibiting Repair Mediated DNA Demethylation

Gemcitabine is a cytotoxic cytidine analog, which is widely used in anti-cancer therapy. One mechanism by which gemcitabine acts is by inhibiting nucleotide excision repair (NER). Recently NER was implicated in Gadd45 mediated DNA demethylation and epigenetic gene activation. Here we analyzed the effect of gemcitabine on DNA demethylation. We find that gemcitabine inhibits specifically Gadd45a mediated reporter gene activation and DNA demethylation, similar to the topoisomerase I inhibitor camptothecin, which also inhibits NER. In contrast, base excision repair inhibitors had no effect on DNA demethylation. In Xenopus oocytes, gemcitabine inhibits DNA repair synthesis accompanying demethylation of oct4. In mammalian cells, gemcitabine induces DNA hypermethylation and silencing of MLH1. The results indicate that gemcitabine induces epigenetic gene silencing by inhibiting repair mediated DNA demethylation. Thus, gemcitabine can function epigenetically and provides a tool to manipulate DNA methylation

Helix–hairpin–helix protein MJ1434 from Methanocaldococcus jannaschii and EndoIV homologue TTC0482 from Thermus thermophilus HB27 do not process DNA uracil residues

The 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 Δung-strains as hosts for production of putatively DNA-U processing enzymes provides a simple safeguard

DNA uracil repair initiated by the archaeal ExoIII homologue Mth212 via direct strand incision

No genes for any of the known uracil DNA glycosylases of the UDG superfamily are present in the genome of Methanothermobacter thermautotrophicus ΔH, making it difficult to imagine how DNA-U repair might be initiated in this organism. Recently, Mth212, the ExoIII homologue of M. thermautotrophicus ΔH has been characterized as a DNA uridine endonuclease, which suggested the possibility of a novel endonucleolytic entry mechanism for DNA uracil repair. With no system of genetic experimentation available, the problem was approached biochemically. Assays of DNA uracil repair in vitro, promoted by crude cellular extracts, provide unequivocal confirmation that this mechanism does indeed operate in M. thermautotrophicus ΔH

DNA uracil repair initiated by the archaeal ExoIII homologue Mth212 via direct strand incision

No genes for any of the known uracil DNA glycosylases of the UDG superfamily are present in the genome of Methanothermobacter thermautotrophicus ΔH, making it difficult to imagine how DNA-U repair might be initiated in this organism. Recently, Mth212, the ExoIII homologue of M. thermautotrophicus ΔH has been characterized as a DNA uridine endonuclease, which suggested the possibility of a novel endonucleolytic entry mechanism for DNA uracil repair. With no system of genetic experimentation available, the problem was approached biochemically. Assays of DNA uracil repair in vitro, promoted by crude cellular extracts, provide unequivocal confirmation that this mechanism does indeed operate in M. thermautotrophicus ΔH

The Methanothermobacter thermautotrophicus ExoIII homologue Mth212 is a DNA uridine endonuclease

The genome of Methanothermobacter thermautotrophicus, as a hitherto unique case, is apparently devoid of genes coding for general uracil DNA glycosylases, the universal mediators of base excision repair following hydrolytic deamination of DNA cytosine residues. We have now identified protein Mth212, a member of the ExoIII family of nucleases, as a possible initiator of DNA uracil repair in this organism. This enzyme, in addition to bearing all the enzymological hallmarks of an ExoIII homologue, is a DNA uridine endonuclease (U-endo) that nicks double-stranded DNA at the 5′-side of a 2′-d-uridine residue, irrespective of the nature of the opposing nucleotide. This type of activity has not been described before; it is absent from the ExoIII homologues of Escherichia coli, Homo sapiens and Methanosarcina mazei, all of which are equipped with uracil DNA repair glycosylases. The U-endo activity of Mth212 is served by the same catalytic center as its AP-endo activity

A new discovered repair mechanism for hydrolytically damaged DNA cytosine residues, established in the thermophilic archaeon Methanothermobacter thermautotrophicus ΔH

Die DNA-Basen Cytosin, Adenin und Guanin können an den exozyklischen Aminogrupen durch Hydrolyse spontan desaminiert werden. Die Cytosin-Desaminierung führt zur RNA-Base Uracil und ist von besonderer mutagener Bedeutung, da sie (i), relativ zur Desaminierung anderer Basen, häufig stattfindet und (ii) unrepariert zu C/G- nach T/A-Transitionsmutationen in der Hälfte der Nachkommenschaft einer Zelle führt. Die Initiation einer vom Sequenzkontext unabhängigen (generellen) Reparatur von Uracilresten in DNA war bislang allein auf Uracil-DNA-Glykosylasen zurückzuführen. Diese Enzyme leiten einen weit verbreiteten Mechanismus der DNA-Reparatur, die Basen-Exzisions-Reparatur, ein. Bei einigen Organismen ist jedoch bislang keine generelle Uracil-DNA-Glykosylase entdeckt worden. DNA thermophiler Organismen ist aus fundamentalen Gründen einem erhöhten Risiko an Mutationen ausgesetzt. Aus diesem Grund ist es erstaunlich, dass beim thermophilen Archaeon Methanothermobacter thermautotrophicus ΔH bislang keine generell agierende Uracil-DNA-Glykosylase entdeckt worden ist. In vorliegender Arbeit konnte nach Fraktionierung einer Zellmasse vonM. thermautotrophicus ΔH eine Uracil prozessierende Aktivität nachgewiesen werden. Bei dieser Aktivität handelte es sich nicht um eine Glykosylase-, sondern eine Endonuklease-Aktivität an Uridinresten in DNA. Über massenspektrometrische Analyse, Selektion und Überprüfung geeigneter Kandidaten, konnte dem Genprodukt des Leserahmens mth212 diese Aktivität zugeordnet werden. Das Enzym, das bisher nur als Homolog der ExoIII-Familie von AP-Endonukleasen beschrieben worden war, ist zusätzlich zu den bekannten Aktivitäten von ExoIII-Enzymen in der Lage, Strangbrüche auf der 5‘ Seite eines Uridinrestes in doppelsträngiger DNA unabhängig vom gegenüberliegenden Nukleotid und unabhängig vom Sequenzkontext zu setzen. Durch diesen Strangbruch werden 3‘-OH und 5‘-Phosphat-Termini von dem Enzym erzeugt. Mit einem Proteinextrakt von M. thermautotrophicus ΔH wurde eine effiziente Reparatur einer U/G-Fehlpaarung (dem Produkt der Cytosin-Desaminierung in doppelsträngiger DNA) beobachtet. Die Initiation dieser Reparatur konnte über gezielte Adsorbtion von Mth212 an eine anti-Mth212-Antikörpermatrix fast vollständig verhindert werden. Die Reparatur der Fehlpaarung wurde demnach durch die neu entdeckte DNA-Uridin-Endonuklease eingeleitet. Die Enzyme DNA-Uridin-Endonuklease (Mth212), DNA-Polymerase B, 5‘ Flap-Endonuklease und DNA-Ligase aus M. thermautotrophicus ΔH wurden heterolog in E. coli produziert, bis nahe an die Homogenität gereinigt und die enzymatischen Aktivitäten in Einzelnachweisen bestätigt. Eine Rekonstitution der DNA-Uridin-Reparatur konnte mit diesem Satz an Enzymen durchgeführt werden. Dabei war jedoch der Anteil an ligierten Reparaturintermediaten (vollständige Reparatur) nur zu einem deutlich geringeren Anteil als mit dem Proteinextrakt detektierbar. Das könnte an der Auswahl bestimmter Enzyme, vor allem der DNA Polymerase, oder an einem Fehlen von Reparaturfaktoren gelegen haben

GADD45a physically and functionally interacts with TET1

AbstractDNA demethylation plays a central role during development and in adult physiology. Different mechanisms of active DNA demethylation have been established. For example, Growth Arrest and DNA Damage 45-(GADD45) and Ten-Eleven-Translocation (TET) proteins act in active DNA demethylation but their functional relationship is unresolved. Here we show that GADD45a physically interacts – and functionally cooperates with TET1 in methylcytosine (mC) processing. In reporter demethylation GADD45a requires endogenous TET1 and conversely TET1 requires GADD45a. On GADD45a target genes TET1 hyperinduces 5-hydroxymethylcytosine (hmC) in the presence of GADD45a, while 5-formyl-(fC) and 5-carboxylcytosine (caC) are reduced. Likewise, in global analysis GADD45a positively regulates TET1 mediated mC oxidation and enhances fC/caC removal. Our data suggest a dual function of GADD45a in oxidative DNA demethylation, to promote directly or indirectly TET1 activity and to enhance subsequent fC/caC removal