Mechanisms Influencing Topological Integrity of the Chromatin During Replication Stress and DNA Double Strand Break

Sanjeevamurthy, Chaithra (2024). Mechanisms Influencing Topological Integrity of the Chromatin During Replication Stress and DNA Double Strand Break. Doctor of Philosophy (PhD) thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.00096528

Abstract

DNA unwinding during replication and transcription generates topological constraints, which are resolved by DNA topoisomerases. Changes in chromatin supercoiling have been shown to impact various cellular processes, including replication, transcription, formation of non-B DNA cruciform structures, DNA-protein interactions, and higher-order chromatin structure. However, the mechanisms and pathways involved in preventing aberrant topological outcomes during DNA damage remain unclear. In this study, we utilized the yeast Saccharomyces cerevisiae as a model system to investigate the topological state of replicating chromosomes in Rad53-deficient cells under conditions of replication stress. By mapping the genome-wide supercoiling of chromatin, we made several interesting observations. Firstly, we found that Rad53 plays a role in regulating the negative supercoiling of chromatin. In Rad53-deficient cells, there was a decrease in negative supercoiling at gene boundaries, RNA polymerase I, II, and III coding regions, transposable elements, telomeres, and replication origins. Interestingly, the rad53-K227A mutant exhibited increased positive supercoiling at replication origins. Additionally, we observed unregulated binding of Top1 and Top2 on the chromatin in the rad53-K227A mutant.

Based on these findings, we propose that the increased positive supercoiling generated at replication origins in the rad53-K227A mutant leads to the formation of fork reversal. Consequently, topological alterations occur, resulting in decreased genome-wide accumulation of RNA-DNA hybrids and eviction of nucleosome core particles histone H3. Moreover, the rad53-K227A mutant exhibited increased phosphorylation of γH2A at replication origins, possibly due to replication fork associated DNA damage.

In parallel, we investigated chromatin supercoiling in wild-type strains with induced single DNA double-strand breaks (DSBs) mediated by the HO endonuclease. We observed an increase in negative supercoiling around the DNA DSB on chromosome III, and this effect was dependent on the activation of the checkpoint protein Rad53.