| Project Detail |
DNA replication stress, defined as the slowing or stalling of replication fork progression, is a major source of genomic instability in the early stages of tumorigenesis and is also a common feature of cancer cells. Accumulating evidence suggests that this phenomenon results primarily from head-on conflicts between transcription and replication machineries. Replication fork stalling induced by transcription is caused by co-transcriptional R-loops generated by annealing of the nascent transcript to the template DNA strand behind the transcription complex and is associated with replication fork reversal catalyzed by specific DNA translocases. Our recent work has shown that RECQ1-mediated reverse branch migration on the resulting four-way DNA structures triggers the restart of semiconservative DNA replication, which requires RECQ5 DNA helicase, MUS81/EME1 endonuclease, the DNA ligase IV(LIG4)/XRCC4 complex, the transcription elongation factor ELL and active RNA synthesis. Our results suggested a model for transcription-replication conflict (TRC) resolution wherein fork cleavage-religation cycles catalyzed by MUS81/EME1 and LIG4/XRCC4 relieve the torsional stress in the DNA template generated by TRC allowing ELL-dependent bypass of the transcription complex across the replication-stalling site and subsequent replication restart. However, many questions remain to be answered regarding the molecular mechanisms of R-loop-mediated fork stalling and subsequent reactivation of DNA synthesis. In this project, we aim to provide a clear understanding of the causes of transcription-dependent replication stress, focusing on the role of elevated levels of reactive oxygen species (ROS), which are often present in cancer cells due to altered metabolism or hypoxia, and are also generated by cancer chemotherapeutics such as mitomycin C or cisplatin. Specifically, we will investigate whether ROS are the cause of transcription-dependent replication fork stalling induced by abrogation of the MUS81-LIG4-ELL axis in cancer cells and whether this phenomenon can also be triggered by hypoxia. Moreover, we will explore whether transcription-dependent replication stress is induced by ROS-generating drugs as suggested by our preliminary experiments. In a second line of research, we will elucidate how R-loop-forming transcription complexes halt replication fork progression. Particularly, we will test the model where fork stalling results from the build-up of positive DNA supercoiling between the converging transcription and replication machineries, which is potentiated by the formation of an R-loop. We will also address the question of whether the replicative DNA helicase remains on the DNA during fork stalling at R-loops to nucleate replisome assembly following R-loop removal. Finally, in a third line of research, we aim to gain further insight into the molecular mechanisms underlying TRC resolution via the MUS81-LIG4-ELL axis. We have found that MUS81-initiated restart of R-loop-stalled forks requires Senataxin, an RNA/DNA helicase whose mutations are associated with two progressive neurological disorders termed ataxia oculomotor apraxia 2 (AOA2) and amyotrophic lateral sclerosis 4 (ALS4). Here, we aim to investigate whether Senataxin exerts this function through its helicase activity and interaction with nuclear exosome, which are disrupted by missense mutations found in AOA2 patients. In addition, we will investigate whether the replication restart process at R-loops depends on the physical interactions of RECQ5 with RNA polymerases I and II, which were identified in our previous studies. Our approach combines state-of-the-art methods of molecular and cell biology with advanced microscopy techniques and a broad range of biochemical methods including mass spectrometry-based proteomic profiling. It is anticipated that this research will advance understanding of the basic DNA-repair processes and may open up new avenues for the treatment of cancer. |
