Non-canonical DNA/RNA structures during Transcription-Coupled Double-Strand Break Repair: Roadblocks or Bona fide repair intermediates?

Abstract

Although long overlooked, it is now well understood that DNA does not systematically assemble into a canonical double helix, known as B-DNA, throughout the entire genome but can also accommodate other structures including DNA hairpins, G-quadruplexes and RNA:DNA hybrids. Notably, these non-canonical DNA structures form preferentially at transcriptionally active loci. Acting as replication roadblocks and being targeted by multiple machineries, these structures weaken the genome and render it prone to damage, including DNA double-strand breaks (DSB). In addition, secondary structures also further accumulate upon DSB formation. Here we discuss the potential functions of pre-existing or de novo formed nucleic acid structures, as bona fide repair intermediates or repair roadblocks, especially during Transcription-Coupled DNA Double-Strand Break repair (TC-DSBR), and provide an update on the specialized protein complexes displaying the ability to remove these structures to safeguard genome integrity.

Keywords: Chromatin; DNA double-strand break repair; G-quadruplex; R-loop; RNA:DNA hybrid; Transcription.

Figure 1.. Repression of transcription at damage-associated loci.

Undamaged chromatin is decorated by histone modifications that promote transcription. Upon DSB formation, chromatin is highly modified and participates along with pre-existing marks to facilitate repressive chromatin and altered RNA Pol II, which collectively tune down transcription to coordinate repair activities within these damage gene loci. DSB – DNA double-strand break.

Figure 2.. Transcription-coupled DSB Repair pathway (TC-DSBR) repairs DSBs occurring in transcriptionally active loci.

R-loops and G4 structures accumulate at transcribed genes, mainly over TSS and promoter regions, where they can induce DSBs, resulting in a rapid transcriptional arrest of the local elongating RNA polymerases (RNA Pol II) (see Fig. 1). We propose that secondary DNA structures may inhibit fast and accurate NHEJ pathway, consequently triggering DNA ends processing, further stimulated by CtIP recruitment through LEDGF/H3K36me3 interaction. In G1 cells, this short range resection may delay repair and promote DSB clustering [117]. These processed DNA ends may require DNA Polα dependent fill-in to complete repair by NHEJ. In S/G2 cells, long range resection of the 5’ strand allows formation of a 3’ nucleoprotein filament further available for HR repair using the available sister chromatid as a template.

Figure 3.. Hypotheses for RNA:DNA hybrids and/or R-loops formation at transcriptionally active broken loci.

(i) The signaling of a DSB occurring in a Pol II-bound locus induces transcriptional pausing known to trigger R-loops formation. (ii) Nearby pre-mRNA hybridizes with the single-strand DNA following resection. (iii) Pol II is de novo recruited and uses the single-strand DNA available following resection as a template to generate a RNA:DNA hybrid.

Figure 4.. A proposed model for RNA production around DSB.

A. Genome browser screenshots showing Pol II occupancy (ChIP-seq data in undamaged DIvA cells [129]) and NET-seq data from [127] in undamaged U2OS cells (NET-seq -DSB) and in damaged DIvA cells (NET−seq +DSB), at different scales, around two AsiSI-induced DSBs. The DSB position is indicated with a vertical dotted line. Note that Pol II-embedded transcripts are not exactly starting at the DSB position even following damage. B. A proposed model of RNA:DNA hybrids and R-loops accumulation after DSB occurring in transcriptionally active genes. Top panel: In normal conditions (no DSB), sense and antisense transcription can produce R-loops on melted promoters [17] (see for instance Fig. 4A, 4kb scale, NET-seq data in absence of damage, in blue, where low level of antisense transcription is detected upstream TSS). Downstream of the TSS, elongating Pol II can also produce transient RNA:DNA hybrids during the process of transcription. Lower panel: As a consequence of DSB induction (damaged locus), the transition from initiation to elongation is inhibited. Non elongating Pol II accumulate at melted promoters of damaged genes, increasing sense and antisense transcription (see for example Fig. 4A, at 4kb and 400bp scale, the NET-seq data in presence of DSB, in red), further enhancing R-loops. Moreover, downstream TSS, DSB-induced Pol II pausing, or simply a decrease in Pol II elongation rate, triggers RNA:DNA hybrids accumulation, which may also be further stabilized as the resection machinery uncovers single-strand DNA.

Figure 5.. Possible functions of RNA:DNA hybrids during DSB repair.

RNA:DNA hybrids formed around the break could serve: 1. as a platform to recruit DSB repair proteins; 2. to produce DNA damage response RNAs (DDRNAs); 3. as a template for a RNA-mediated repair mechanism 4. to control resection and single-strand binding proteins assembly, thereby regulating downstream events such as Rad51 nucleofilament assembly and homologous recombination.

Figure 6:. Pre-existing and DSB-induced G4 and RNA:DNA hybrids shall be dissolved for repair completion.

Right side of the DSB: RNA:DNA hybrids that pre-exist and accumulate following DSB shall be removed to ensure RPA loading. SETX, XPG and RNAse H participate in hybrids dissolution. In this respect, RNAse H recruitment by RPA may allow processive hybrids unwinding and RPA spreading. Left side of the DSB: G4 shall also be disassembled for repair. Pif1 is necessary for DNA double-strand G4 unwinding, allowing resection. On another hand, G4 were also proposed to form on the resected strand which may require BLM and PARP3 for dissolution and RPA binding. Together these processes allows Rad51 nucleofilament assembly and HR.