title: Investigating the interplay of replication, transcription, and BER for the induction of DSBs after oxidative stress
Creators: name: Pfeffel, Daniel
Publisher: name: UNSPECIFIED
Year: 2022

description: Every day, a variety of different deoxyribonucleic acid (DNA) lesions occur in our genome, threatening the integrity and inheritance of genetic information. The lesions that occur are diverse and may have distinct consequences. The damage to DNA is thereby often caused by reactive oxygen species (ROS). These can be generated both endogenously and exogenously and can be responsible for the occurrence of various lesions. One of the most common DNA damage related to ROS is oxidative base modification. Several hundreds of these occur spontaneously in our genome every day and require efficient and correct repair via base excision repair (BER). However, several studies in recent years have demonstrated that the repair process of modified bases, and in particular the most abundant oxidative base modification 8-oxoguanine (8-oxoG), appears to be involved in the formation of the most dangerous lesion, the DNA double-strand break (DSB). In this work, the DSB inducing effect of oxidative stress was investigated by treatment with the established oxidative agent H2O2. A cell cycle-specific analysis was performed, which demonstrated the induction of DSBs mainly in the synthesis phase (S phase) of the cell cycle. These breaks were almost exclusively repaired via the homologous recombination (HR) repair pathway, and thus, the emergence of replication-associated one-ended DSBs was assumed. Inhibition of replication thereby led to a significant reduction of DSBs in S phase and confirmed their dependence on the replication process. In addition, a comparable reduction in induced DSBs was demonstrated by inhibition of transcription. Follow-up studies using the proximity ligation assay (PLA) confirmed the occurrence of replication-transcription conflicts after treatment with H2O2, thus identifying these events as the reason for the formation of DSBs. Replication-transcription conflicts usually occur when obstacles in the DNA interfere with the progression of one or both of these two processes. H2O2 treatment induces, to a large extent, the oxidative base modification 8-oxoG which can be removed by the BER mechanism and replaced with a correct base via the formation of a single-strand break (SSB). This work demonstrates that the initiation of BER by the specific 8-oxoguanine glycosylase (OGG1) is required for DSB formation after H2O2 treatment. Inhibition of this initiation resulted in decreased DSB numbers, which in contrast greatly increased if the repair of SSBs was prevented. The removal of the damaged base and the resulting SSB are the reason for the occurence of conflicts between replication and transcription. Due to the involvement of both replication and transcription in the formation of DSBs after H2O2 treatment, break induction should only be able to occur in regions with active transcription. As confirmed by sequencing results in this work, the breaks induced by H2O2 occur more frequently in regions of elevated transcription. In addition, an increased density of genes and GC content could be detected for these regions. This shows that the breaks induced by H2O2 occur in gene-rich regions with strong transcription. Furthermore, it could be observed that this process of DSB induction also takes place without H2O2 treatment in unbertubed, proliferating cells. Thus, it is assumed that this process is significantly involved in the induction of endogenous breaks. A mechanism was elaborated specifically occuring in S phase, requiring the interplay of replication, transcription, as well as BER. This mechanism can explain the mutagenic effect of ROS, which plays an important role in the development of various diseases and clarifies the significance of simple base modifications for the fate of a cell.