Looping out of control: R-loops in transcription-replication conflict

Abstract

Transcription-replication conflict is a major cause of replication stress that arises when replication forks collide with the transcription machinery. Replication fork stalling at sites of transcription compromises chromosome replication fidelity and can induce DNA damage with potentially deleterious consequences for genome stability and organismal health. The block to DNA replication by the transcription machinery is complex and can involve stalled or elongating RNA polymerases, promoter-bound transcription factor complexes, or DNA topology constraints. In addition, studies over the past two decades have identified co-transcriptional R-loops as a major source for impairment of DNA replication forks at active genes. However, how R-loops impede DNA replication at the molecular level is incompletely understood. Current evidence suggests that RNA:DNA hybrids, DNA secondary structures, stalled RNA polymerases, and condensed chromatin states associated with R-loops contribute to the of fork progression. Moreover, since both R-loops and replication forks are intrinsically asymmetric structures, the outcome of R-loop-replisome collisions is influenced by collision orientation. Collectively, the data suggest that the impact of R-loops on DNA replication is highly dependent on their specific structural composition. Here, we will summarize our current understanding of the molecular basis for R-loop-induced replication fork progression defects.

Keywords: DNA replication; Genome stability; R-loops; RNA polymerase; Transcription.

Figure 1.. Co-directional (CD) and head-on (HO) collisions between replisomes and co-transcriptional R-loops in bacteria and eukaryotes.

Parental DNA strands are indicated in black, nascent leading and lagging strands are in red and blue, respectively. Dashed arrows represent directions of replication and transcription.

Figure 2:. R-loop-independent blocks to replication fork progression during transcription-replication conflict.

a, Steric inhibition of fork progression by RNA polymerase. b, Steric inhibition of fork progression promoter-bound transcription factor complexes. c, Fork stalling due to positive torsional strain accumulating between converging replication and transcription machineries. d, Fork stalling due to active fork reversal mediated by fork remodeling enzymes.

Figure 3.. Consequences of CD R-loop-replisome collisions in eukaryotes.

a, CMG unwinding of RNA:DNA hybrids featuring RNA with 5’ flap. b, CMG translocation across RNA:DNA hybrid featuring annealed RNA 5’ end followed by mRNA takeover-mediated leading strand restart. c, Replisome stalling at RNA:DNA hybrids containing secondary structure, such as G4 in DNA or RNA.

Figure 4.. Outcomes of CD R-loop-replisome collisions in bacteria.

a, CD collisions between replisomes and short naked R-loops allow continued replisome progression and uninterrupted leading strand synthesis. b, Replisome bypass of long naked R-loops or RNAP-associated R-loops can involve mRNA takeover or primase-mediated leading strand restart.

Figure 5.. Consequences of HO R-loop-replisome collisions in eukaryotes and bacteria.

a, Bypass and retention of RNA:DNA hybrid on lagging strand at eukaryotic replication fork. b, Unwinding of RNA:DNA hybrid featuring RNA with unannealed 3’ end by replicative DNA helicase in bacteria. c, Bacterial replisome bypass of short RNA:DNA hybrid featuring RNA with annealed 3’ end followed by RNA displacement by replicative DNA polymerase. d, Bacterial replisome bypass of long RNA:DNA hybrid featuring RNA with annealed 3’ end accompanied by retention of RNA:DNA hybrid in replicated daughter strand.

Figure 6.. Bacterial replisome collisions with RNAP-associated R-loops.

a, Short R-loops associated with RNAP result in mRNA takeover-mediated leading strand restart in CD orientation. b, Long RNAP-associated R-loops cause primase-mediated leading strand restart in CD orientation. c, HO RNA-associated R-loop are strong blocks to fork progression.

Figure 7:. DNA Secondary structures exacerbate consequences of R-loop-replisome collisions in eukaryotes.

a, G4s in the displaced ssDNA loop on lagging strand cause gaps in lagging strand. b, Replisome stalling at G4s in displaced ssDNA loop on leading strand. c, Replisome bypass and leading strand restart at G4s in displaced ssDNA loop on leading strand.

Figure 8:. Effect of chromatin on R-loop-replisome collisions in eukaryotes.

a, Replisome stalling at Yra1-stabilized R-loop. b, Replisome stalling at R-loop-associated condensed chromatin.