The roles of RNA in DNA double-strand break repair

Abstract

Effective DNA repair is essential for cell survival: a failure to correctly repair damage leads to the accumulation of mutations and is the driving force for carcinogenesis. Multiple pathways have evolved to protect against both intrinsic and extrinsic genotoxic events, and recent developments have highlighted an unforeseen critical role for RNA in ensuring genome stability. It is currently unclear exactly how RNA molecules participate in the repair pathways, although many models have been proposed and it is possible that RNA acts in diverse ways to facilitate DNA repair. A number of well-documented DNA repair factors have been described to have RNA-binding capacities and, moreover, screens investigating DNA-damage repair mechanisms have identified RNA-binding proteins as a major group of novel factors involved in DNA repair. In this review, we integrate some of these datasets to identify commonalities that might highlight novel and interesting factors for future investigations. This emerging role for RNA opens up a new dimension in the field of DNA repair; we discuss its impact on our current understanding of DNA repair processes and consider how it might influence cancer progression.

Fig. 1

Schematic diagram of the double-stranded break (DSB) repair cascade. Initial recognition of the break recruits the phosphatidylinositol 3-kinase-like kinases (PIKKs), resulting in its activation by autophosphorylation. Activated PIKKs then phosphorylates early participants of the cascade, H2AX and MDC1. The ubiquitin ligase RNF8 recognises and is recruited to phosphorylated MDC1 at the break site, initiating the ubiquitylation of histone H1, which subsequently recruits RNF168 to ubiquitylate H2A. The modified chromatin acts as a marker for the competitive recruitment of either 53BP1 or BRCA1, which facilitate either non-homologous end-joining (NHEJ) or homologous recombination (HR), respectively. NHEJ repair is dependent on the recruitment of the DNA–PK complex (Ku70–80 bound to DNA–PKcs) and end-processing factors such as XRCC4 and Lig4. HR is dependent on resection of the broken DNA to produce a single-stranded DNA overhang that gets coated in a RAD51 filament and invades the sister-chromatid to facilitate templated repair.

Fig. 2

Meta-analysis of proteomic studies into DNA-damage-dependent changes. Venn diagram (left) shows the significant hits shared by each of the studies. Gene ontology slim biological process enrichment analysis (right) shows which gene groups are enriched in the combined list of significant hits from all experiments; the bars represent the fold enrichment of each group. a Three studies that investigated proteins that localise/bind to damaged chromatin: association with chromatin in response to UV damage; co-localisation with γH2AX upon laser micro-irradiation; and in vitro association with an RPA-ssDNA construct. b Three studies that investigated post-translational modification changes in response to damage: ubiquitylation changes in response to UV damage; PARylation changes in response to a variety of genotoxic agents; and phosphorylation changes up to 1 h post 6 Gy of radiation. c Dataset overlap of the combined studies from (a) and the combined studies from (b) with the RNA interactome.

Fig. 3

Structure of R-loops and their formation behind RNA polymerases. Partially unannealed double-stranded DNA allows complimentary single-stranded RNA to anneal to one of the free DNA strands. This often occurs behind transcription bubbles, due to the DNA being in an unwound state with the complimentary RNA being actively synthesised and therefore held in close proximity to the DNA.

Fig. 4

Schematic model of the possible mechanisms of RNA-dependent DNA repair (RDDR). Repair is initiated in the same way as canonical double-stranded break (DSB) repair; however, at the point of the ubiquitylation cascade, an RNA production/processing step occurs, which produces DDRNAs. This DDRNA either takes the form of small RNA, which could hybridise to the broken DNA via the aid of helicases such as DDX1 and DHX9, or of a long RNA molecule, which could bridge the break by hybridising to the DNA via a RAD52-dependent strand invasion mechanism. Either the small RNA acts as a sequence-specific signal and allows propagation of canonical DSB repair pathways, or the long RNA molecule is used as a template for high-fidelity repair.