Ribonucleotides in DNA - Application in genome-wide DNA polymerase tracking and physiological role in eukaryotes

Abstract

The genetic code in the eukaryotic cell is stored in the form of DNA, which is more resistant to hydrolysis than RNA. Replication fidelity and DNA repair mechanisms are in place to ensure genomic integrity to preserve the information encoded. Despite DNA polymerases’ discrimination against ribonucleotides, they are frequently incorporated into DNA and even in the presence of efficient ribonucleotide removal pathways, ribonucleotides may remain stably incorporated in the DNA. Ribonucleotides can be used as a marker of DNA replication enzymology by using HydEn-seq, a next-generation sequencing technique for the genome-wide mapping of ribonucleotides. I aimed to elucidate the activities of the specialized translesion synthesis DNA polymerase η in yeast. By using a steric gate variant that incorporates more ribonucleotides and by tracking those ribonucleotides, I determined a lagging strand preference dependent on its C-terminus in Paper I. The findings suggest a possible extension of the ‘division of labor’ among replicative polymerases to the specialized polymerases. Moreover, I was interested in the physiological role of incorporated ribonucleotides and used an extension of the HydEn-seq method outlined in Paper II, to map and quantitate ribonucleotides simultaneously. By investigating ribonucleotide incorporation into mouse mitochondrial DNA (mtDNA) in Paper III, we found that ribonucleotides are acquired mostly up until adulthood and are not connected to age-related mtDNA instability, suggesting relatively good tolerance of incorporated ribonucleotides in mtDNA. To gain a more comprehensive view on incorporated ribonucleotides in the DNA of mammals, I mapped and quantitated incorporated ribonucleotides in nuclear DNA (nDNA) and mtDNA from murine blood, bone marrow, brain, heart, kidney, liver, lung, muscle and spleen in Paper IV. I found tissue-dependent variations in the number and the identity of incorporated ribonucleotides and marked differences between nDNA and mtDNA. The ribonucleotide distribution in both types of DNA was non-random and in nDNA affected by the proximity of genomic features, which in most cases increased the number of embedded ribonucleotides locally as compared to random positions in the nDNA. The thesis extends the knowledge of DNA polymerase η’s activity and the physiological role that incorporated ribonucleotides play in DNA. This more detailed characterization of the incorporated ribonucleotides genome-wide is a basic requirement for the understanding of diseases associated with genome instability, such as certain types of cancers or Aicardi-Goutières syndrome.