Interplay of DNA replication, repair and chromatin under radiation stress

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.