An R-loop-initiated CSB–RAD52–POLD3 pathway suppresses ROS-induced telomeric DNA breaks

Abstract

Reactive oxygen species (ROS) inflict multiple types of lesions in DNA, threatening genomic integrity. How cells respond to ROS-induced DNA damage at telomeres is still largely unknown. Here, we show that ROS-induced DNA damage at telomeres triggers R-loop accumulation in a TERRA- and TRF2-dependent manner. Both ROS-induced single- and double-strand DNA breaks (SSBs and DSBs) contribute to R-loop induction, promoting the localization of CSB and RAD52 to damaged telomeres. RAD52 is recruited to telomeric R-loops through its interactions with both CSB and DNA:RNA hybrids. Both CSB and RAD52 are required for the efficient repair of ROS-induced telomeric DSBs. The function of RAD52 in telomere repair is dependent on its ability to bind and recruit POLD3, a protein critical for break-induced DNA replication (BIR). Thus, ROS-induced telomeric R-loops promote repair of telomeric DSBs through CSB–RAD52–POLD3-mediated BIR, a previously unknown pathway protecting telomeres from ROS. ROS-induced telomeric SSBs may not only give rise to DSBs indirectly, but also promote DSB repair by inducing R-loops, revealing an unexpected interplay between distinct ROS-induced DNA lesions.

Alternative lengthening of telomeres: from molecular mechanisms to therapeutic outlooks

“…For example, loss of TRF1 increases telomere fragility, which may trigger ALT [92]. TRF2 is required for the induction of R-loops at telomeres after oxidative DNA damage, and it may also promote ALT by increasing telomeric R-loops [93]. Both TRF1 and TRF2 are SUMOylated by MMS21, allowing them to localize to APBs [94].…”

The DNA secondary structures at telomeres and genome instability

“…Then, several studies also confirm the function of R-loop in telomere maintenance [69,70]. Our group recently reported that the R-loop-CSB-RAD52-POLD3 axis contributes to the repair of Reactive oxygen species (ROS) induced telomeric damage in ALT cancer [8]. Target R-loop interacting proteins at genome are also possible to enhance the cell killing effects.…”

“…1). Telomeric DNA are TTA GGG tandem repeats, which are susceptible for oxidative DNA damage and hotspot regions for formation of DNA secondary structures such as t-loop, D-loop, G-quadruplex (G4), and R-loop [6][7][8]. Single strand G-rich overhang folds back and invades into the double-stranded telomere tract to form a T-loop and D-loop structure, protecting the end of chromosome from being recognize as double strands breaks (DSBs) [9][10][11].…”

“…R-loops are the three-stranded nucleic acid structure that contains a DNA: RNA hybrid and displaced DNA strand. We recently discovered that ROS-induced DNA damage at telomeres triggers R-loop accumulation in a TERRA and TRF2-dependent manner [8].…”

Is there a role of phase partitioning in coordinating DNA damage response?

“…Rad52 has also been demonstrated to lead to transcript-dependent DSB repair (19,20). This perspective is supported by the mounting evidence accounting for R-loops as physiological regulators in the genome (21,22). Notably, Rad52 (in yeast) and FUS (in human) were observed to contribute to the formation of a molecular biocondensate at DSB sites, carrying out different roles, namely the organization of nuclear microtubule filaments (23), protecting the resected end of lesions and promoting DNAdamage signaling (24,25), as well as recruiting other DSB-repair-related enzymes (26).…”