Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.

Integrative Genomics Viewer

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.

/pdf/topoisomerase-1-prevents-replication-stress-at-r-loop-50edaqrtpx.pdf

Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites.

Walking a tightrope: The complex balancing act of R-loops in genome stability.

https://www.zora.uzh.ch/id/eprint/201549/1/The_Hammer.pdf

The Hammer and the Dance of Cell Cycle Control.

Identifying how R-loops are generated is crucial to know how transcription compromises genome integrity. We show by genome-wide analysis of conditional yeast mutants that the THO transcription complex, prevents R-loop formation in G1 and S-phase, whereas the Sen1 DNA-RNA helicase prevents them only in S-phase. Interestingly, damage accumulates asymmetrically downstream of the replication fork in sen1 cells but symmetrically in the hpr1 THO mutant. Our results indicate that: R-loops form co-transcriptionally independently of DNA replication; that THO is a general and cell-cycle independent safeguard against R-loops, and that Sen1, in contrast to previously believed, is an S-phase-specific R-loop resolvase. These conclusions have important implications for the mechanism of R-loop formation and the role of other factors reported to affect on R-loop homeostasis.

/pdf/harmful-r-loops-are-prevented-via-different-cell-cycle-552b8iy1qf.pdf

Harmful R-loops are prevented via different cell cycle-specific mechanisms.

Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts.

Conflicts between replication and transcription machineries have profound effects on chromosome duplication, genome organization, as well as evolution across species. Head-on conflicts (lagging strand genes) are significantly more detrimental than co-directional conflicts (leading strand genes). The source of this fundamental difference is unknown. Here, we report that topological stress underlies this difference. We find that head-on conflict resolution requires the relaxation of positive supercoils by type II topoisomerases, DNA gyrase and Topo IV. Interestingly, we find that after positive supercoil resolution gyrase also introduces excessive negative supercoils at head-on conflict regions, driving pervasive R-loop formation. The formation of these R-Loops through gyrase activity is most likely caused by the diffusion of excessive negative supercoils through RNA polymerase spinning. Altogether, these results provide critical mechanistic insights into head-on replication-transcription conflicts and suggest that gyrase plays a fundamental role in the evolution of head-on genes.

/pdf/topological-stress-is-responsible-for-the-detrimental-2gstg6ty1h.pdf

Kevin S. Lang

Papers

Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts.

Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts