Collisions between the DNA replication and transcription machineries (replication-transcription conflicts) appear to be common in eukaryotic cells. Although these conflicts have long been studied as a potential source of DNA damage and, therefore, a threat to genome integrity, we lack a detailed molecular understanding of how the presence of transcribing RNA polymerases on DNA affects the progress of replication, and of the mechanism(s) by which these replication-transcription conflicts give rise to DNA damage. Another consequence of transcription during DNA replication is elevated levels of ribonucleoside triphosphates (rNTPS) ? the substrate for RNA polymerases. Due to incomplete discrimination between rNTPs and dNTPs by the replicative DNA polymerases, large numbers of ribonucleotides are mis-incorporated into the genome during each round of replication; this burden is estimated at > 1 million ribonucleotides per cell division in human cells, making ribonucleotides by far the most abundant lesion in eukaryotic DNA. Mis-incorporated ribonucleotides are removed via the ribonucleotide excision repair (RER) pathway, and impaired removal is linked to several human diseases. However, it is not known how ribonucleotides impact chromatin ? the higher-order structure of DNA ? or conversely how chromatin affects RER. Furthermore, it has not been determined whether all ribonucleotides are equally amenable to repair or what may underlie differences in RER efficiency through the genome. The proposed work encompasses two ongoing projects: The first project addresses how orientation-dependent effects on replication progression and genome integrity arise at transcribed genes. To achieve this, we use a recently developed quantitative method to assay the movement of the replisome genome-wide at high resolution, in combination with genome-wide interrogation of DNA double-strand break formation and a novel assay to map nascent DNA strands in the context of an arrested replication fork. The second project uses a combination of genome-wide assays and in vitro biochemistry to delineate how ribonucleotides destabilize nucleosomes (the basic repeating unit of chromatin), how nucleosomes affect RER initiation by the RNase H2 enzyme, and to elucidate the dynamics of RER at all loci in the genome. The machineries responsible for DNA replication, transcription, and DNA repair are highly conserved throughout eukaryotes. Both projects will be carried out in the budding yeast Saccharomyces cerevisiae: the small genome, rapid replication, and genetic manipulability of S. cerevisiae make this an ideal model in which to study the intersection of fundamental biological processes. Therefore, the results of this work will provide molecular insights into genome instability in humans, and will be directly applicable to our understanding of the etiology and progression of cancer as well as rare diseases including Aicardi-Goutires syndrome.
Simultaneous replication and transcription of the same DNA template leads to collisions between the replication and transcription machineries, and to the incorporation of large numbers of ribonucleotides into the genome ? both of which may be a significant source of DNA damage in eukaryotes. One project will characterize the mechanisms through which two nuclear RNA polymerases responsible for transcription ? RNAP2 and RNAP3 ? impede replication, and the molecular basis for DNA damage at conflict sites. The other project investigates how ribonucleotides mis-incorporated during DNA replication impact genome structure and function, and how they are repaired.
