Mechanism of efficient double-strand break repair by a long non-coding RNA

Abstract

Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1’s ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA–RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy.

New Faces of old Friends: Emerging new Roles of RNA-Binding Proteins in the DNA Double-Strand Break Response

“…Indeed, numerous lncRNAs have been shown to directly bind to SWI/SNF chromatin remodeling complexes to modulate their activities in response to DNA damage (Prensner et al, 2013;Cajigas et al, 2015;Adriaens et al, 2016). In addition, KU and DNA-PKcs are themselves RBPs and their RNA-binding activity is important for efficient NHEJ-mediated DSB repair (Yoo and Dynan, 1998;Baltz et al, 2012;Britton et al, 2013;Thapar et al, 2020). An RNA that is especially relevant in this context is LINP1 (lncRNA in nonhomologous end joining pathway 1), which directly binds to KU80 and stabilizes the KU-DNA-PKcs complex at DSB ends (Zhang et al, 2016;.…”

Assessing G4-Binding Ligands In Vitro and in Cellulo Using Dimeric Carbocyanine Dye Displacement Assay

“…While it is true that G-quadruplexes have a positive peak at 210 nm, this greatly depends on the buffer conditions used. Notably, a recent report of G-quadruplex in LINP1 lncRNA was characterized using CD did not display a 210 nm peak albeit folding into parallel G-quadruplex [ 45 ]. B6,5 does not alter the secondary G-quadruplex structure adopted by RNA ( Supplementary Figure S6 ).…”

X‐ray scattering reveals disordered linkers and dynamic interfaces in complexes and mechanisms for DNA double‐strand break repair impacting cell and cancer biology

“…When KU is bound to DNA‐PKcs to form DNA‐PK assembly, the KU80CTR region is far more extended from the KU core (~60 Å) 85 , 87 than in the free state as identified by SAXS (Figure 1c ). 2 , 50 The C‐terminal helix of KU80CTR is even more distant (~80 Å) from the KU core. 75 , 85 , 87 Thus, the KU80CTR domain, including the KU80CTR C‐terminus, must undergo a large displacement during KU interaction with DNA‐PKcs.…”

“…We find that SAXS is an enabling technique to structurally characterize protein conformations in solution under near‐physiological conditions with high‐throughput and super‐resolution. 48 , 49 Fortunately, collecting SAXS data are straightforward and essentially available to any scientist who has protein, RNA, or DNA 1 , 4 , 8 , 23 , 50 due to the availability of synchrotron beamline facilities such as SIBYLS. 51 Importantly, SAXS results readily complement and enhance structural results from cryo‐EM, MX, NMR, and computational modeling, so we see SAXS as a premier technique for integrative structural biology.…”

“… 70 Importantly this presynaptic complex protects and holds two DNA ends in concert with core scaffold proteins XRCC4‐XLF and LigIV plus PAXX (PAralog of XRCC4 and XLF), which can be functionally replaced by lncRNA (long noncoding RNA) LINP1 in NHEJ. 50 Together the DNA‐PK complex, XRCC4‐XLF scaffold proteins, and LigIV form the long‐range (LR) complex, as the two DNA ends are protected but not processed or aligned. Further DNA end processing can be required to remove damaged DNA and non‐ligatable end groups at the termini of the DSB to facilitate ligation.…”