A dynamic cellular and molecular analysis of R-loop mediated transcription replication conflicts

• Applicants: Dr. Stephan Hamperl; Professor Brian Luke, Ph.D.
• Subject Area: General Genetics and Functional Genome Biology ; Biochemistry ; Cell Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 552129721

Transcription-replication conflicts (TRCs) constitute a major intrinsic source of genome instability, which is a hallmark of cancer cells. In a conflict scenario the two machineries may encounter each other either in a head-on (HO) or co-directional (CoD) orientation. TRCs that involve R-loop structures, three-stranded nucleic acid structures containing RNA:DNA hybrids show particular detrimental effects on genome stability in different cellular systems including yeast and mammalian cells. Despite extensive studies on TRCs in a variety of organisms, we have very limited knowledge regarding the molecular events taking place right at the conflict region where a replication fork encounters an R-loop containing transcription complex. Recently, we have developed a microscopy-based assay for the locus specific simultaneous monitoring of replication and transcription in budding yeast. Our unpublished data indicate that this system can be harnessed to monitor TRCs at an R-loop prone sequence that exhibits distinct molecular features compared to non-R loop forming transcription unit. In parallel, we have developed and applied an approach for the comprehensive analysis of site-specific chromatin structure and posttranslational modification state, as well as unique tools for the analysis of R-loops at telomeric regions as another example of a TRC site involving R-loops. We now plan to combine these methodologies to obtain an in-depth characterization of the molecular mechanisms of R-loop mediated TRCs. Our collaborative effort will allow us to shed new light on the dynamics of R-loop formation during replisome progression and the effects of R-loops on replication progression and transcription dynamics. In addition, we will obtain novel information on how TRCs involving R-loops affect the chromatin composition and accessibility. Finally, application of these approaches for the study of telomeric R-loops will allow us to reveal novel regulatory mechanisms for these unique genomic regions. Overall, we expect our work to provide essential mechanistic knowledge on the genetic and epigenetic principles of how transcription and replication are regulated and coordinated at R-loop forming genes to prevent harmful TRCs.

• DFG Programme: Research Grants
• International Connection: Israel
• International Co-Applicant: Professor Amir Aharoni, Ph.D.