Single-molecule imaging reveals replication fork coupled formation of G-quadruplex structures hinders local replication stress signaling

Abstract

Guanine-rich DNA sequences occur throughout the human genome and can transiently form G-quadruplex (G4) structures that may obstruct DNA replication, leading to genomic instability. Here, we apply multi-color single-molecule localization microscopy (SMLM) coupled with robust data-mining algorithms to quantitatively visualize replication fork (RF)-coupled formation and spatial-association of endogenous G4s. Using this data, we investigate the effects of G4s on replisome dynamics and organization. We show that a small fraction of active replication forks spontaneously form G4s at newly unwound DNA immediately behind the MCM helicase and before nascent DNA synthesis. These G4s locally perturb replisome dynamics and organization by reducing DNA synthesis and limiting the binding of the single-strand DNA-binding protein RPA. We find that the resolution of RF-coupled G4s is mediated by an interplay between RPA and the FANCJ helicase. FANCJ deficiency leads to G4 accumulation, DNA damage at G4-associated replication forks, and silencing of the RPA-mediated replication stress response. Our study provides first-hand evidence of the intrinsic, RF-coupled formation of G4 structures, offering unique mechanistic insights into the interference and regulation of stable G4s at replication forks and their effect on RPA-associated fork signaling and genomic instability.

Impact of G-Quadruplexes and Chronic Inflammation on Genome Instability: Additive Effects during Carcinogenesis

“…These data support computational studies that have suggested more than 350,000 PG4s reside outside of telomeric regions; with the inclusion of non-canonical motifs, the total number of PG4 motifs is over 700,000 in the human genome [50,51]. Recently, single-molecule fluorescence microscopy was combined with unbiased pattern recognition algorithms to analyze G4 structures associated with replication [52]. In U2OS cells, ~2% of replisomes, identified by PCNA and MCM helicase antibodies, colocalize with G4 structures.…”

G-Quadruplexes and Their Ligands: Biophysical Methods to Unravel G-Quadruplex/Ligand Interactions

“…Several reports have described the formation of G4 structures within endogenous chromatin, and their ability to recruit transcription factors to promote active transcription [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. The location of those G4 structures was revealed using an antibody-based G4 chromatin immunoprecipitation sequencing (G4 ChIP–seq) approach [ 21 ], and suggests that they play a crucial role in critical cellular processes such as DNA replication [ 29 , 30 ], DNA damage repair [ 26 ], transcription [ 22 , 23 ], translation [ 31 ] and epigenetic modifications [ 32 ]. By using G4 ChIP–seq, Hänsel-Hertsch et al showed a reduction in the number of detected DNA G4s (10,000) in genome [ 21 ].…”

The Relevance of G-Quadruplexes for DNA Repair

“…The regulatory function of G4 structures is contrasted by their potential to induce genome instability. Depending on the location and the time, the formation of G4s can cause genome instability by altering transcription, causing replication fork stalls or inducing mutations [17,21,[33][34][35][36][37]. To preserve genome stability helicases, ensure the correct unfolding of G4 structures within cells [38].…”