An experimental system to investigate the consequences of co-transcriptional R-loop formation in the context of DNA replication in mammalian cells
Final Report Abstract
Transcription and replication machineries compete for the same DNA template and interference between these two fundamental processes has potential catastrophic outcomes for the genome. Head-on (HO) conflicts destabilize bacterial and yeast genomes to a greater extent than co-directional (CD) collisions, but this has not been shown in human cells. Genome stability is also compromised by R-loops, cotranscriptional RNA:DNA hybrid structures with a displaced single-stranded DNA. Collisions between replication forks and R-loop-transcription complexes may contribute to the genomic instability observed in R-loop accumulating cells, but the transient nature of R-loops and the variability of the replication program in human cells prevented a mechanistic dissection of these events. During my research fellowship sponsored by the DFG, I established an episomal system to distinguish between R-loop and non R-loopmediated HO and CD transcription-replication conflicts in human cells. The results indicate that both HO and CD collisions may cause DNA breaks and induce distinct DNA damage responses in the cell. Unexpectedly, my results also identify the replisome as a regulator of RNA:DNA hybrid levels depending on the orientation to transcription: HO collisions impair replication fork progression, activate the ATR-Chk1 pathway, and result in high levels of R-loops. Conversely, the level of hybrids is significantly reduced upon collision in CD orientation, suggesting that the replisome can resolve RNA:DNA hybrids when formed on the leading strand. This link between R-loop levels and the replication program is supported by our finding that replication stress induced by inhibitors or deregulation of origin firing leads to increased levels of genomic R-loops. This finding is consistent with a model that unperturbed replication of the human genome is biased towards CD collisions leading to reduced levels of R-loops and genome stability. Under replication stress, I speculate that the number of HO collision events increases, leading to the observed accumulation of genomic R-loops. I continued working on this project during a third year (funded by Karlene Cimprich). During that time, I completed the data set and finalized a first mansucript on the episomal system which will be submitted for publication soon. I further characterized the direct structural and physical consequences of R-loop mediated transcription-replication conflicts to the underlying DNA template. This includes observation of DNA breaks on the plasmid constructs, analysis of replication intermediates by 2D gel electrophoresis, and the identification of mutational signatures after R-loop mediated transcriptionreplication conflicts by next-generation sequencing. I plan to focus on the further characterization of this highly tractable system as it has a high potential to provide insight into many interesting biological questions at the interface of transcription, R-loop formation and DNA replication. A system, which would not exist without the support from the research fellowship.
Publications
- (2014). The contribution of co-transcriptional RNA:DNA hybrid structures to DNA damage and genome instability. DNA Repair (Amst.) 19, 84–94
Hamperl, S., and Cimprich, K.A.
(See online at https://doi.org/10.1016/j.dnarep.2014.03.023) - (2016) Conflict resolution in the genome: how transcription and replication make it work. Cell, Volume 167, Issue 6, 1 December 2016, Pages 1455-1467
Hamperl, S., Cimprich, K.A.
(See online at https://doi.org/10.1016/j.cell.2016.09.053) - Transcription-Replication Conflict Orientation Modulates R-Loop Levels and Activates Distinct DNA Damage Responses. Cell, Volume 170, Issue 4, 10 August 2017, Pages 774-786.e19
Hamperl, S., Bocek, Michael J., Saldivar, J.C., Swigut, T. and Cimprich, K.A.
(See online at https://doi.org/10.1016/j.cell.2017.07.043)
