Consequences and Resolution of Transcription-Replication Conflicts

Abstract

Transcription-replication conflicts occur when the two critical cellular machineries responsible for gene expression and genome duplication collide with each other on the same genomic location. Although both prokaryotic and eukaryotic cells have evolved multiple mechanisms to coordinate these processes on individual chromosomes, it is now clear that conflicts can arise due to aberrant transcription regulation and premature proliferation, leading to DNA replication stress and genomic instability. As both are considered hallmarks of aging and human diseases such as cancer, understanding the cellular consequences of conflicts is of paramount importance. In this article, we summarize our current knowledge on where and when collisions occur and how these encounters affect the genome and chromatin landscape of cells. Finally, we conclude with the different cellular pathways and multiple mechanisms that cells have put in place at conflict sites to ensure the resolution of conflicts and accurate genome duplication.

Keywords: G-MiDS; MIDAS; R-loops; chromatin; common fragile sites; early replicating fragile sites; fork reversal; genomic instability; replication stress; torsional stress; transcription–replication conflicts.

Figure 1

Coordination of transcription and replication over the cell cycle. In G1-phase, transcription (orange) is temporally separated from replication (blue), allowing for redistribution of replication origins from actively transcribed gene bodies, thereby assuring improved spatial coordination of the two machineries in the subsequent S-phase. TRCs are most likely to occur in early S-phase cells when significant overlap between active transcription and replication sites exists. Mid- and late S-phase stage cells show improved segregation of the nuclear transcription (orange) and replication (blue) foci. However, certain TSSs remain under-replicated and require G2/M DNA synthesis (G/MiDS) to complete genome replication. Finally, difficult-to-replicate regions, such as common fragile sites (CFS), rely on mitotic DNA synthesis (MIDAS) to complete genome duplication and ensure genomic stability during mitosis.

Figure 2

Consequences of TRCs on genome stability as studied by reporter systems. (A) In bacterial cells, the use of reporter genes such as LacZ, thyP3 or luxABCDE, in the CD or HO orientation can be used to induce TRCs and study their mutational outcome. Even though HO-oriented conflicts are more deleterious, both orientations induce R-loops and mutation of gene promoter and coding sequences. Additionally, insertions and deletions (indels) are more abundant at CD-TRCs, preferentially at the promoter region. (B) In the model organism S. cerevisiae, LEU2 reporter constructs were engineered in the CD or HO orientation to study the recombinogenic outcome of TRCs. In addition, the integration of an inducible LEU2 or LYS2 gene in the CD or HO orientation to a known replication origin allows studying the genetic outcome of TRCs in the chromosomal context. HO-oriented TRCs showed a stronger R-loop-dependent induction of recombination as well as a higher frequency of indels, frameshift mutations and DNA damage. (C) In mammalian cells, the cloning of either the mAIRN gene (R-loop prone) or of the ECFP gene (without R-loop formation) in a vector either in the CD or HO orientation relative to a viral unidirectional replication origin (oriP) allows discriminating between R-loop-prone and non-R-loop HO- or CD-TRCs. HO-oriented TRCs show persistent R-loop formation and activation of the ATR kinase, while CD-oriented TRCs show low levels of R-loop formation and signaling via the ATM kinase.

Figure 3

(A) In bacterial chromosomes, replication forks are frequently impeded by HO transcription and highly expressed CD transcription units such as the ribosomal RNA gene cluster (rrn). HO encounters typically lead to more severe consequences such as cell death and DNA damage repair response, but also CD encounters require replication restart factors to complete genome duplication. (B) Left panel: Arising TRCs at ERFS could lead to prolonged stalling of transcription and replication, favoring R-loop formation and DNA damage that can cause chromosome breakage and ERFS instability. Right panel: Transcribing RNA polymerases shift the position of replication origins on cellular DNA. This misplacement leads to origin paucity and long-distance traveling replication forks in the late S-phase that cause under-replicated DNA regions responsible for CFS fragility. (C) Global perturbations of replication and transcription dynamics can lead to imbalance of the two processes and globally higher transcription–replication interference.

Figure 4

Pathways used to resolve a transcription–replication conflict. (A) When faced with a transcriptional block, the replisome (in blue) can skip the RNAP (in orange) and reprime downstream using the PRIM1 primase when the block is on the lagging strand, or the PRIMPOL1 polymerase when the block is on the leading strand. (B) In case of a CD conflict, the replisome can also displace the RNAP and use the hybridized RNA as a primer to reinitiate replication. (C) Numerous pathways exist to simply remove and, in some cases, degrade the RNAP and the potential associated R-loops from the chromatin to allow continuous DNA synthesis. (D) In case of persistent RNAP complexes, the replication fork can undergo fork reversal to stabilize the fork and give time to resolve the conflict by the above-mentioned mechanisms. (E) The reversed fork can also undergo a cycle of fork cleavage and re-ligation. This mechanism uses RECQ5 to inhibit fork reversal and the endonuclease MUS81 to cleave the fork. This relieves torsional stress, and the fork can then be re-ligated by RAD52 and LIG4 that in turn allows the resumption of transcription and the removal of the replisome block.