RADX Sustains POT1 Function at Telomeres to Counteract RAD51 Binding, which Triggers Telomere Fragility

Abstract

The 3' terminal DNA extensions at chromosome ends can become engaged into multiple biochemical reactions during DNA replication, telomerase-mediated telomere extension, homology directed DNA repair, nucleolytic processing and DNA damage checkpoint activation. To keep these activities in check, telomeric 3' overhangs can be hidden in t-loop structures or they associate with specialized proteins such as POT1. Here, we explore the telomeric microenvironment using a proximity-dependent labeling approach and identify the oligonucleotide/oligosaccharide-binding (OB)-fold containing protein RADX. RADX binds single-stranded telomeric DNA throughout the cell cycle along with POT1, suppressing accumulation of fragile telomeres, which are indicative of telomere replication defects.Telomere fragility in POT1 and RADX double-depleted cells was due to accumulation of the RAD51 recombinase at telomeres. RADX also supports DNA damage signaling at POT1depleted telomeres counteracting RAD51 binding. Thus, RADX represents next to POT1 a second OB-fold containing single-strand telomere binding protein sustaining telomere protection.

RADX condenses single-stranded DNA to antagonize RAD51 loading

“…These studies suggest that RADX antagonizes RAD51 at replication forks to balance fork remodeling and stabilization to maintain genome stability ( 17 ). Intriguingly, a recent study also showed that RADX is involved in telomere protection ( 19 ). RADX binds exposed single-stranded telomeric DNA along with POT1 to antagonize the accumulation of RAD51 and reduce sister telomere associations.…”

“…RADX-depleted cells exhibit excessive RAD51 activity and illegitimate recombination, suggesting that RADX is a negative regulator of RAD51 that functions at replication forks to maintain genome stability ( 17 ). A recent study also showed that RADX is involved in telomere maintenance by binding single-stranded telomeric DNA along with POT1 to antagonize RAD51 ( 19 ). The mechanistic basis of how RADX acts on ssDNA to negatively regulate RAD51 is unclear.…”

The Human Telomeric Proteome During Telomere Replication

“…HEK 293E cl75 was generated by transfecting pSpCas9(BB)-2A-Puro (PX459) plasmid expressing gRNAs targeting the genomic region proximal to the start codon of TRF1 (TRF1-gRNA: 5’-GCGAGCCATTTAACATGGCGG-3’) or TRF2 (TRF2-gRNA: 5’-TCTATCATGGCCGCGGGAGC-3’) and non-linearized repair template plasmid 67 . The repair template contained 600bp homology region flanking both sites of the start codon of TRF1 or TRF2 amplified from genomic DNA of HEK293E cells, and combined with three tandemly repeated FLAG tags, EcoRI restriction site, Kozak sequence and linker base pairs by using overlapping PCR.…”

RADX condenses single-stranded DNA to antagonize RAD51 loading

“…RADX binds exposed single telomeric DNA along with POT1 to antagonize the accum RAD51 and reduce sister telomere associations. Depletion RAD51 or BRCA2 at telomeres rescued RADX depletion, s that RADX also antagonizes homologous recombination in th (19).…”

“…Loss of RADX leads to excessive RAD51 activity replication forks, slowing elongation and causing fork collap studies suggest that RADX antagonizes RAD51 at replicatio balance fork remodeling and stabilization to maintain genom (17). Intriguingly, a recent study also showed that RADX is in telomere protection (19). RADX binds exposed single telomeric DNA along with POT1 to antagonize the accum RAD51 and reduce sister telomere associations.…”