Einfluss von DNA-Basenmodifikationen auf die Doppelhelixstabilität und Genomaktivität
Biochemie
Zellbiologie
Zusammenfassung der Projektergebnisse
It is well known that, besides the four canonical DNA bases, the mammalian genome harbors several chemically modified bases that diversify the genome. The predominant DNA modification in the mammalian genome is cytosine methylation (5mC) and its generation is catalyzed by DNA methyltransferases (Dnmts). This cytosine derivative can further be modified by the Tet enzyme protein family, resulting in the oxidized forms 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). While DNA base modifications are known to be essential for many cellular processes, such as differentiation and development, their influence on DNA physical properties and the ensuing effects on genome metabolism, are still poorly understood. Hence, we focused on the interplay of cytosine modifications and DNA metabolic processes. We, therefore, established cellular systems to modulate the extent of cytosine variants, based on changing the expression levels of modification writer enzymes, as well as based on transfection of modified nucleotides into cells. We find highest duplex stabilities for methylated DNA (5mC) in vitro and in vivo. This effect is reverted by all oxidized cytosine variants. Accordingly, in the presence of the destabilizing 5-hydroxymethylcytosine (5hmC), we find higher DNA transcription and replication rates in in vitro reactions using well-defined reconstituted systems as well as in in vivo analyses within the complex organization of the cell. Similarly, we show faster helicase-mediated DNA unwinding in the presence of oxidized and in the absence of methylated cytosines. We additionally investigated the properties of the mouse embryonic stem (mES) cell replicon, i.e. the DNA replicated from one origin, and the number of active replication origins. By combining genome-wide origin mapping, single-molecule and super-resolution microscopy (3D-SIM), we demonstrate that each replication focus visualized by 3D-SIM, corresponds to an individual replicon, and up to one quarter represents unidirectional replication forks. Furthermore, mES cell replicons are smaller than in somatic cells, ensuing from the activation of twice as many origins spaced at half the distance. Based on this in-depth characterization of DNA replication in wildtype mES cells, we showed that Dnmt triple knock-out mES cells pass more rapidly through S-phase stage II when highly methylated DNA is replicated and, consistently, have faster replication forks. These observations are not the result of altered chromatin condensation, structure, accessibility, or histone marks and we conclude that the absence of 5mC enhances DNA unwinding and consequently DNA replication fork speed. We, therefore, propose that variegated effects of modified cytosines on DNA helix stability may constitute a mechanism for local fine-tuning of DNA processes. In summary, our data contribute to a better understanding of different levels of epigenetic regulation involved in nuclear metabolic processes.
Projektbezogene Publikationen (Auswahl)
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Developmental differences in genome replication program and origin activation. Nucleic Acids Research, 48(22), 12751-12777.
Rausch, Cathia; Weber, Patrick; Prorok, Paulina; Hörl, David; Maiser, Andreas; Lehmkuhl, Anne; Chagin, Vadim O.; Casas-Delucchi, Corella S.; Leonhardt, Heinrich & Cardoso, M. Cristina
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DNA Modification Readers and Writers and Their Interplay. Journal of Molecular Biology, 432(6), 1731-1746.
Rausch, Cathia; Hastert, Florian D. & Cardoso, M. Cristina
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Cytosine base modifications regulate DNA duplex stability and metabolism. Nucleic Acids Research, 49(22), 12870-12894.
Rausch, Cathia; Zhang, Peng; Casas-Delucchi, Corella S.; Daiß, Julia L.; Engel, Christoph; Coster, Gideon; Hastert, Florian D.; Weber, Patrick & Cardoso, M. Cristina
