R‐loop formation during S phase is restricted by PrimPol‐mediated repriming

Abstract

During DNA replication, conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R‐loops, three‐stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single‐stranded DNA, are important drivers of damage arising from such conflicts. How R‐loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood. Here, we show in vivo how R‐loop formation drives a short purine‐rich repeat, (GAA)10, to become a replication impediment that engages the repriming activity of the primase‐polymerase PrimPol. Further, the absence of PrimPol leads to significantly increased R‐loop formation around this repeat during S phase. We extend this observation by showing that PrimPol suppresses R‐loop formation in genes harbouring secondary structure‐forming sequences, exemplified by G quadruplex and H‐DNA motifs, across the genome in both avian and human cells. Thus, R‐loops promote the creation of replication blocks at susceptible structure‐forming sequences, while PrimPol‐dependent repriming limits the extent of unscheduled R‐loop formation at these sequences, mitigating their impact on replication.

Timeless couples G quadruplex detection with processing by DDX11 during DNA replication

“…When unwound, certain sequences, often repetitive or of low complexity, can adopt a variety of non-B form structures, including hairpins, cruciforms, triplexes and quadruplexes 1 . It is becoming clear that secondary structure formation is a frequent event during replication, even at genomically abundant sequences previously thought not to be a major source of difficulty 2 . To prevent such sequences causing havoc with the genetic and epigenetic stability of the genome, cells deploy an intricate network of activities to counteract secondary structure formation and limit its effects.…”

“…These activities include proteins that bind and destabilise DNA structures and specialised helicases that unwind them 3 . In addition, the repriming activity of PrimPol can be deployed to confine a structure into a minimal region of single stranded DNA, limiting the potential dangers of exposing extensive ssDNA in a stalled replisome 2,4 .…”

RNase H1 and H2 are differentially regulated to eliminate RNA-DNA hybrids

“…RNase H2 is able to remove RNA-DNA hybrids in R-loops as well as perform RER (Cerritelli and Crouch, 2009). MMS induces DNA damage and replication stress, both of which can lead to the stabilization and generation of R-loops (Hamperl et al, 2017;Svikovic et al, 2018;Teloni et al, 2018) (Figure 2), which likely contribute to the MMS sensitivity of rnh1 rnh201 cells. R-loop stabilization in sen1-1 mutants is also responsible for lethality in rnh1 rnh201 double mutants (Costantino and Koshland, 2018).…”

The sequence of the extruded non-template strand determines the architecture of R-loops

“…The formation of secondary structures within the extruded non-template strand of R-loops has long been hypothesized, mostly because at least some R-loops were shown to form over nucleotide sequences with structure-forming potential, as determined computationally (7,20,21). But to our knowledge, this possibility had never been rigorously tested experimentally.…”