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Interplay of DNA replication, repair and chromatin under radiation stress

Scholl, Annina :
Interplay of DNA replication, repair and chromatin under radiation stress.
[Online-Edition: http://tuprints.ulb.tu-darmstadt.de/6691]
Technische Universität , Darmstadt
[Dissertation], (2017)

Offizielle URL: http://tuprints.ulb.tu-darmstadt.de/6691

Kurzbeschreibung (Abstract)

The human genome contains around 1.2% protein coding sequences and over 50% repetitive elements. Within the nucleus, it is packed together with proteins and RNAs and is highly organized as chromatin. The lowest organization level is the DNA double helix wrapped around the core histones to form the nucleosome, multiple nucleosomes form the beads on a string. Higher order chromatin structures on the Mbp range have been termed the 1 Mbp domain or topologically associated domains (TADs), but what lies in between the beads on a string and the higher order structures is not fully understood. DNA maintenance processes like DNA replication and repair are dictated by many features of chromatin organization, including structural organization, compaction level and epigenetics. DNA replication and repair structures, such as the phosphorylated form of the histone variant H2AX, have been associated with forms of structural chromatin organization. In the first part of the thesis, DNA replication and repair were utilized to identify a possible basic unit of structural chromatin organization using super-resolution microscopy. Therefore, single replicons were labeled by nucleotide incorporation and the cells irradiated with X-ray radiation and stained for H2AX. Both replicons and repair (nano)foci are compared, considering their DNA content, size and localization. Despite their independent distribution and the accompanied low probability of colocalization we were able to identify a surprisingly high number of colocalizing replicons and repair (nano)foci. These were assumed to be most comparable, since they are likely localized on the same chromatin structure. Direct comparison revealed that the majority of foci exhibited an astonishing similarity, suggesting that replicons and repair (nano)foci might indeed be based on the same basic unit of chromatin structure. This chromatin structure shows sizes between 27.5-91.2 kbp for human and 61.6-118.6 kbp for murine cells. With a possible formation of chromatin loops, this structure can be ranked between the beads on a string and higher order chromatin structures like TADs or the 1 Mbp domain. The sizes are consistent with microscopy based data (40-160 kbp) and comparable to Hi-C data (185 kbp) of chromatin structures. Repetitive elements can be divided into interspersed elements, like the Alu and LINE1 elements, and tandemly repeated DNA like satellite III. While Alu is associated with euchromatin, LINE1 and satellite III are (predominantly) heterochromatic, with satellite III being located in pericentromeric heterochromatin. The second part of the thesis focusses on the replication timing and repair kinetics of these elements. Alu is replicated during early S-phase as expected for a euchromatic element, LINE1 is replicated throughout S-phase, with the majority at the early to mid S-phase transition, while satellite III is replicated exclusively during the mid to late S-phase transition. The repair kinetics of Alu and LINE1 were similar to global DNA repair kinetics, while damage in satellite III persists longer. All of this is compatible with replication and repair kinetics of the respective chromatin types and suggests that repetitive elements are well integrated into the genome, despite their reputation as “junk” DNA.

Typ des Eintrags: Dissertation
Erschienen: 2017
Autor(en): Scholl, Annina
Titel: Interplay of DNA replication, repair and chromatin under radiation stress
Sprache: Englisch
Kurzbeschreibung (Abstract):

The human genome contains around 1.2% protein coding sequences and over 50% repetitive elements. Within the nucleus, it is packed together with proteins and RNAs and is highly organized as chromatin. The lowest organization level is the DNA double helix wrapped around the core histones to form the nucleosome, multiple nucleosomes form the beads on a string. Higher order chromatin structures on the Mbp range have been termed the 1 Mbp domain or topologically associated domains (TADs), but what lies in between the beads on a string and the higher order structures is not fully understood. DNA maintenance processes like DNA replication and repair are dictated by many features of chromatin organization, including structural organization, compaction level and epigenetics. DNA replication and repair structures, such as the phosphorylated form of the histone variant H2AX, have been associated with forms of structural chromatin organization. In the first part of the thesis, DNA replication and repair were utilized to identify a possible basic unit of structural chromatin organization using super-resolution microscopy. Therefore, single replicons were labeled by nucleotide incorporation and the cells irradiated with X-ray radiation and stained for H2AX. Both replicons and repair (nano)foci are compared, considering their DNA content, size and localization. Despite their independent distribution and the accompanied low probability of colocalization we were able to identify a surprisingly high number of colocalizing replicons and repair (nano)foci. These were assumed to be most comparable, since they are likely localized on the same chromatin structure. Direct comparison revealed that the majority of foci exhibited an astonishing similarity, suggesting that replicons and repair (nano)foci might indeed be based on the same basic unit of chromatin structure. This chromatin structure shows sizes between 27.5-91.2 kbp for human and 61.6-118.6 kbp for murine cells. With a possible formation of chromatin loops, this structure can be ranked between the beads on a string and higher order chromatin structures like TADs or the 1 Mbp domain. The sizes are consistent with microscopy based data (40-160 kbp) and comparable to Hi-C data (185 kbp) of chromatin structures. Repetitive elements can be divided into interspersed elements, like the Alu and LINE1 elements, and tandemly repeated DNA like satellite III. While Alu is associated with euchromatin, LINE1 and satellite III are (predominantly) heterochromatic, with satellite III being located in pericentromeric heterochromatin. The second part of the thesis focusses on the replication timing and repair kinetics of these elements. Alu is replicated during early S-phase as expected for a euchromatic element, LINE1 is replicated throughout S-phase, with the majority at the early to mid S-phase transition, while satellite III is replicated exclusively during the mid to late S-phase transition. The repair kinetics of Alu and LINE1 were similar to global DNA repair kinetics, while damage in satellite III persists longer. All of this is compatible with replication and repair kinetics of the respective chromatin types and suggests that repetitive elements are well integrated into the genome, despite their reputation as “junk” DNA.

Ort: Darmstadt
Fachbereich(e)/-gebiet(e): 10 Fachbereich Biologie
10 Fachbereich Biologie > Cell Biology and Epigenetics
Hinterlegungsdatum: 20 Aug 2017 19:55
Offizielle URL: http://tuprints.ulb.tu-darmstadt.de/6691
URN: urn:nbn:de:tuda-tuprints-66913
Gutachter / Prüfer: Cardoso, Prof. Dr. M. Cristina ; Laube, Prof. Dr. Bodo
Datum der Begutachtung bzw. der mündlichen Prüfung / Verteidigung / mdl. Prüfung: 12 Juli 2017
Alternatives oder übersetztes Abstract:
AbstractSprache
Das menschliche Genom besteht zu ca. 1,2% aus Protein-codierenden Sequenzen und zu mehr als 50% aus repetitiven Elementen. Innerhalb des Nukleus ist DNA dicht verpackt mit Proteinen und RNAs und als Chromatin hoch organisiert. Das unterste Organisationslevel ist die DNA Doppelhelix, die sich um ein Histon-Oktamer wickelt und das Nukleosom bildet. Mehrere aneinander gekettete Nukleosomen bilden eine Art Perlenschnur, die beads on a string. Höhere Formen von Chromatin-Struktur wurden beschrieben als 1 Mbp Domäne oder Kontakt-Domänen (TADs) in der Größenordnung von Mbp, aber was zwischen den beads on a string und der 1 Mbp Domäne oder TADs liegt ist bis heute nicht vollständig klar. DNA Replikation und DNA Reparatur werden durch z. B. Chromatin-Organisation, Verdichtung und Epigenetik reguliert. Außerdem wurden Strukturen beider Prozesse, z. B. die phosphorylierte Form der Histon-Variante H2AX, in Verbindung mit struktureller Chromatin-Organisation gebracht. Im ersten Teil dieser Thesis werden Replikation und Reparatur genutzt um eine gemeinsame mögliche Grundeinheit der Chromatin-Struktur zu identifizieren. Dazu wurden einzelne Replikons durch Nukleotid-Inkorporation markiert und Zellen bestrahlt um H2AX zu färben. Trotz ihrer unabhängigen Verteilung und der damit verbundenen geringen Co-lokalisations-Wahrscheinlichkeit waren wir dazu in der Lage eine überraschend hohe Anzahl co-lokalisierender Foci zu identifizieren. Diese sollten am besten miteinander vergleichbar sein, da sie wahrscheinlich auf der gleichen Chromatin-Struktur positioniert sind. Im direkten Vergleich sind sich der größte Teil an Replikons und Reparatur (Nano)Foci erstaunlich ähnlich, was darauf hindeutet, dass beide scheinbar wirklich auf der selben Grundeinheit der Chromatin-Organisation basieren. Diese Grundeinheit hat eine Größe von 27,5-91,2 kbp für menschliche und 61,6-118,6 kbp für murine Zellen, ist möglicherweise in Form von Schlaufen (loops) organisiert und ist zwischen den beads on a string und der 1 Mbp Domäne bzw. TADs anzusiedeln. Die Größen sind konsistent mit bereits beschriebenen Mikroskopie-basierenden (40-160 kbp) und vergleichbar mit Hi-C-basierenden Daten (185 kbp). Repetitive Elemente können in eingestreute (interspersed), wie Alu und LINE1 Elemente, und hintereinander-wiederholte Elemente (tandem repeats), wie Satellit III, unterteilt werden. Alu ist euchromatisch, LINE1 und Satellit III (mehrheitlich) heterochromatisch, wobei es sich bei Satellit III um perizentrisches Heterochromatin handelt. Im zweiten Teil dieser Arbeit wird der Replikations-Zeitpunkt und die Reparatur-Kinetik dieser Elemente untersucht. Alu wird während der frühen, LINE1 während der gesamten S-phase repliziert, wobei der Großteil im Übergang zwischen früher und mittlerer S-phase repliziert wird. Satellit III wird ausschließlich im Übergang zwischen mittlerer und später S-phase repliziert. Die Reparatur-Kinetiken von Alu und LINE1 sind der globalen Reparatur-Kinetik ähnlich, während Schäden in Satellit III länger bestehen bleiben. Diese Beobachtungen sind vereinbar mit denen für die entsprechenden Chromatin-Typen, was darauf hindeutet, dass repetitive Elemente, trotz ihrem Ruf als „Schrott-DNA“, gut in das Genom integriert sind.Deutsch
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