Transcription-Replication Conflicts as a Source of Genome Instability

Abstract

Transcription and replication both require large macromolecular complexes to act on a DNA template, yet these machineries cannot simultaneously act on the same DNA sequence. Conflicts between the replication and transcription machineries (transcription-replication conflicts, or TRCs) are widespread in both prokaryotes and eukaryotes and have the capacity to both cause DNA damage and compromise complete, faithful replication of the genome. This review will highlight recent studies investigating the genomic locations of TRCs and the mechanisms by which they may be prevented, mitigated, or resolved. We address work from both model organisms and mammalian systems but predominantly focus on multicellular eukaryotes owing to the additional complexities inherent in the coordination of replication and transcription in the context of cell type-specific gene expression and higher-order chromatin organization.

Keywords: DNA repair; RNA polymerase; TRCs; genomic instability; origin firing; replication stress; replisome; transcription; transcription–replication conflicts.

Figure 1

Genome organization and transcription-replication collisions (TRCs) across species. A. Codirectional (CD) TRCs occur between the replisome and RNA polymerase (RNAP) oriented in the same direction on leading-strand DNA. CD-TRCs can occur in to permutations: RNAP following the replisome on newly-synthesized DNA (Codirectional TRC – 1), or the replisome following RNAP (Codirectional TRC – 2). Head-on (HO) TRCs occur between the replisome and RNAP oppositely oriented on lagging-strand DNA. Co-transcriptional R-loops, RNA:DNA hybrids, are linked to TRCs. B. Bacterial genomes contain a single replication origin (oriC), and the most highly transcribed and essential genes are oriented codirectionally with oriC (ref). HO-oriented genes accumulate mutations, but are essential for bacterial survival. Specifically, lagging-strand genes are enriched antibiotic resistance and virulence factors. C. Yeast origins occur at sequence-specific ARS sequences, and forks are blocked at the 3’ end of rRNA gene units by Fob1-replication fork barriers (RFBs). Pif1-family helicases are required for progression through programmed RFBs in yeast, and more generally the termini of highly-transcribed gene units such as tRNA genes. D. Replication origins occur preferentially at transcription start sites (TSSs) of long and transcriptionally-active genes in mammalian cells, facilitating codirectional movement through gene bodies. CD-TRCs at TSSs could be endogenous sources of DNA damage and replication stress in murine lymphocyte models (TRIs as in REF, ERFSs as in REF), or regions of mitotic replication (G-MiDS as in REF). G4 quadruplex structures are enriched at TSSs, on lagging strand DNA, and on persistent R-loops. G4s, R-loops, and topological stress are barriers to replication fork progression. Helicases (DNA and RNA:DNA), topoisomerases, and RNases promote fork progression through unprogrammed barriers.

Figure 2

Replication dysregulation causes TRCs A. Short G1 and premature S-phase entry causes increased usage of canonical origins and over-replication leading to double strand breaks (DSBs) resulting from head-to-tail-fork collisions from mixed G1- and S-phase forks. (Pfander ref) Cyclin E1 overexpression causes intragenic origin firing and TRCs linked to fork collapse and genomic rearrangements (Halazonetis ref), as the result of incomplete MCM redistribution by transcription machinery. In contrast, low-dose HU causes dormant origin firing at transcription termination sites of highly-transcribed genes due to MCM clearance of gene bodies by transcription. B. Several pathways of replication fork recovery follow TRCs. Replication fork restart by MUS81 fork cleavage leads to RAD52/LIG4/POLD3-dependent fork religation. Replication fork reversal is triggered by MUS81 cleavage, which activates ATR, which limits excessive MUS81 cleavage. Replication fork repriming past R-loop and G4 secondary structures requires PRIMPOL (primase polymerase). Local RNAP removal by ATAD5, RECQ5, or PNUTS resolves TRCs.

Figure 3

Transcription dysregulation causes TRCs A. Replication and transcription are normally coordinated through several mechanisms (discussed in section 2). Hypertranscription by oncogenes (HRAS, B-catenin), estradiol, or BET inhibitors leads to hypertranscription that causes replication stress and TRCs. MYCN-low neuroblastoma cancer cells are prone to TRCs through defective recruitment of Aurora kinase and the nuclear exosome to transcribed gene units, resulting in antisense transcription, genomic instability, and cancer cell death. B. Senataxin (SETX), yeast homolog Sen1, guards against TRCs through multiple mechanisms: SETX and BRCA1 resolve R-loops at transcription termination sites (TTSs); Sen1 removes RNAP at TRCs in yeast; SETX prevents against FANCD2- and MUS81-mediated fork cleavage and mitotic DNA synthesis (MiDAS). C. Transcription on nascent DNA promotes transfer of epigenetic histone marks, which encourages site-specific origin firing in subsequent cell cycles.