BRCA1 binds TERRA RNA and suppresses R-Loop-based telomeric DNA damage.

R-loop structures act as modulators of physiological processes such as transcription termination, gene regulation, and DNA repair. However, they can cause transcription-replication conflicts and give rise to genomic instability, particularly at telomeres, which are prone to forming DNA secondary structures. Here, we demonstrate that BRCA1 binds TERRA RNA, directly and physically via its N-terminal nuclear localization sequence, as well as telomere-specific shelterin proteins in an R-loop-, and a cell cycle-dependent manner. R-loop-driven BRCA1 binding to CpG-rich TERRA promoters represses TERRA transcription, prevents TERRA R-loop-associated damage, and promotes its repair, likely in association with SETX and XRN2. BRCA1 depletion upregulates TERRA expression, leading to overly abundant TERRA R-loops, telomeric replication stress, and signs of telomeric aberrancy. Moreover, BRCA1 mutations within the TERRA-binding region lead to an excess of TERRA-associated R-loops and telomeric abnormalities. Thus, normal BRCA1/TERRA binding suppresses telomere-centered genome instability.

Effect of the Spiroiminodihydantoin Lesion on Nucleosome Stability and Positioning.

DNA is constantly under attack by oxidants, generating a variety of potentially mutagenic covalently modified species, including oxidized guanine base products. One such product is spiroiminodihydantoin (Sp), a chiral, propeller-shaped lesion that strongly destabilizes the DNA helix in its vicinity. Despite its unusual shape and thermodynamic effect on double-stranded DNA structure, DNA duplexes containing the Sp lesion form stable nucleosomes upon being incubated with histone octamers. Indeed, among six different combinations of lesion location and stereochemistry, only two duplexes display a diminished ability to form nucleosomes, and these only by ∼25%; the other four are statistically indistinguishable from the control. Nonetheless, kinetic factors also play a role: when the histone proteins have less time during assembly of the core particle to sample both lesion-containing and normal DNA strands, they are more likely to bind the Sp lesion DNA than during slower assembly processes that better approximate thermodynamic equilibrium. Using DNase I footprinting and molecular modeling, we discovered that the Sp lesion causes only a small perturbation (±1-2 bp) on the translational position of the DNA within the nucleosome. Each diastereomeric pair of lesions has the same effect on nucleosome positioning, but lesions placed at different locations behave differently, illustrating that the location of the lesion and not its shape serves as the primary determinant of the most stable DNA orientation.