Ribonucleotides and chromatin - a genomics approach

• Applicant: Dr. Vanessa Kellner
• Subject Area: General Genetics and Functional Genome Biology ; Cell Biology
• Term: from 2019 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 433825641

The misincorporation of ribonucleotides instead of deoxyribonucleotides by DNA polymerases is a frequent error during DNA replication and leads to the accumulation of up to one million genomic ribonucleotide monophosphates (rNMPs) per human cell division. If they remain unrepaired, rNMPs cause DNA replication stress and genome instability. The conserved RNase H2 enzyme initiates error-free ribonucleotide excision repair (RER) to allow removal of the rNMP and sealing of the created single-stranded DNA break. Deficiencies in RER lead to an accumulation of genomic rNMPs. RNase H2 mutations were identified to be an underlying cause of Aicardi-Goutières syndrome (AGS), a severe autoinflammatory and neurological disorder. Moreover, recent findings link a loss of RNase H2 activity to cancer. Mechanistically, RER is well-defined, and seems to be restricted to a post-replicative phase of the cell cycle, when DNA is packed into chromatin. How chromatin structure influences RER efficiency is poorly characterized. Nucleosomes could interfere with efficient repair by sterically blocking RNase H2. In addition, rNMPs have been demonstrated in vitro to directly affect both DNA structure and DNA-protein interactions. Thus, chromatin structure and cellular transcription should be impacted in vivo by unrepaired rNMPs. Importantly, these could be so far neglected factors contributing to the pathology of AGS and RNase H2-deficient cancers. Previous work mainly analyzed bulk effects of loss of RNase H2 and focused on the direct impact of rNMPs on genome integrity. Locus-specific information and RER kinetics throughout the cell cycle are not available to date. Additionally, the impact of chromatin on RER and the effect of unrepaired rNMPs on chromatin-related processes such as transcription and nucleosome assembly remain to be defined. The goal of my proposed research project is to elucidate the temporal and spatial regulation of RER genome wide. To this end, I will employ hydrolytic end-sequencing, a deep-sequencing method co-developed by my host that can map rNMPs in the Saccharomyces cerevisiae genome at single nucleotide-level. Firstly, I will determine both overall repair efficiency and locus-specific differences in RER throughout the cell cycle, as well as the impact of chromatin on RER. Secondly, I will use RNA-seq and in vitro repair assays to address the effect of unrepaired rNMPS on chromatin structure and transcription. Finally, I will define the molecular basis of transcription-dependent removal of rNMPs, a potential novel repair pathway suggested by our preliminary data. In summary, this work will elucidate the interconnection between chromatin and RER and contribute novel insights into the pathology of AGS and cancer.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Duncan Smith, Ph.D.