Transcription-mediated replication hindrance: a major driver of genome instability

Abstract

Genome replication involves dealing with obstacles that can result from DNA damage but also from chromatin alterations, topological stress, tightly bound proteins or non-B DNA structures such as R loops. Experimental evidence reveals that an engaged transcription machinery at the DNA can either enhance such obstacles or be an obstacle itself. Thus, transcription can become a potentially hazardous process promoting localized replication fork hindrance and stress, which would ultimately cause genome instability, a hallmark of cancer cells. Understanding the causes behind transcription-replication conflicts as well as how the cell resolves them to sustain genome integrity is the aim of this review.

Keywords: DNA–RNA hybrids; chromosome fragility; genetic instability; replication fork stalling; transcription.

Figure 1.

Replication fork progression and obstacles. (A) A simplified version of replication forks moving away from a replication origin. Replisomes contain the CMG (MCMs, Cdc45, and GINS) replicative helicase, polymerases α, δ, and ε, and a plethora of additional factors that ensure fork progression, such as histone chaperones (as exemplified by FACT) and remodelers. (B) Obstacles to replication fork progression. Fork progression can be hampered by topological stress (panel i); certain chromatin structures such as heterochromatin (panel ii); other nonnucleosomal DNA-bound proteins (panel iii), as exemplified by the Tus protein in bacteria or FOB1-mediated fork barriers in the yeast rDNA; DNA damage, ranging from single-strand breaks (SSBs) and DSBs to interstrand cross-links (ICLs) or base modifications (panel iv); non-B DNA structures, including G quadruplexes (G4), hairpins, DNA–RNA hybrids, and R loops as well triplex or cruciform nucleic acid structures that can contain DNA and RNA (panel v); and the transcription machinery itself (panel vi).

Figure 2.

Transcription and its potential to stall replication. (A) The RNAPII transcription cycle. RNAPII at its pre-initiation stage sits on DNA with GTFs waiting to be activated by TFIIH. Once activated, elongating RNAPII is ready to synthetize the RNA with the help of TEFs. The RNA is then cotranscriptionally processed into an export-competent mRNP, with gene gating facilitating transcription–export coupling. During elongation, RNAPII pauses at regulatory regions and can even backtrack. Once terminated, RNAPII is released from the DNA. (B) Transcription-induced obstacles. In addition to the transcription machinery itself, which is bound to DNA and could block fork progression, transcription enhances the occurrence of structures that hamper replication fork progression. Transcription elongation causes accumulation of positive supercoiling ahead of and negative supercoiling behind the RNAP, enhances the probability of DNA damage, or can promote the formation of non-B DNA structures such as G4 or DNA–RNA hybrids, which have been associated with chromatin compaction.

Figure 3.

Head-on versus codirectional T–R conflicts. Transcription and replication machineries can encounter each other when travelling in head-on (panel i) or codirectional (panel ii) orientation with different consequences for the cell. Whereas the RNAP embraces both DNA strands and can constitute an obstacle by itself in both orientations, other transcription-derived obstacles such as supercoiling or DNA–RNA hybrids will have different effects depending on the orientation of the conflict. (Panel i) Head-on T–R conflicts might be enhanced by the generation of positive supercoiling in front of both machineries, whether they are stabilized or not by the presence of a blocked RNAP and/or DNA–RNA hybrids. (Panel ii) Codirectional T–R conflicts are known to be less deleterious. Although the negative supercoiling accumulated behind RNAP might facilitate the formation of DNA–RNA hybrids, these would likely be dissolved by the replication fork given the 3′–5′ polarity of the eukaryotic replicative helicase.

Figure 4.

Cellular response to T–R conflicts. (A) The fate of a replication fork facing a transcription-induced obstacle would likely depend on the nature of the block, whether affecting only one or both replicating strands. (B) Possible outcomes after T–R conflicts. Repriming ahead of the fork can directly solve encounters of the lagging strand with obstacles, such as DNA–RNA hybrids. In contrast, blocks in unwinding, such as those caused by head-on T–R conflicts might induce fork reversal. Fork arrest, possibly involving some uncoupling and long ssDNA accumulation, triggers the activation of the checkpoint, which is responsible for maintaining the stability of forks, thus preventing irreversible collapse. Replication-associated repair functions, such as FACT, BRCA1, BRCA2, and FA repair pathway, are required for proper fork progression through T–R conflicts such as R loops, likely with the help of specialized helicases.