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

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.

Cancer-associated mutations in the iron-sulfur domain of FANCJ affect G-quadruplex metabolism.

FANCJ/BRIP1 is an iron-sulfur (FeS) cluster-binding DNA helicase involved in DNA inter-strand cross-link (ICL) repair and G-quadruplex (G4) metabolism. Mutations in FANCJ are associated with Fanconi anemia and an increased risk for developing breast and ovarian cancer. Several cancer-associated mutations are located in the FeS domain of FANCJ, but how they affect FeS cluster binding and/or FANCJ activity has remained mostly unclear. Here we show that the FeS cluster is indispensable for FANCJ's ability to unwind DNA substrates in vitro and to provide cellular resistance to agents that induce ICLs. Moreover, we find that FANCJ requires an intact FeS cluster for its ability to unfold G4 structures on the DNA template in a primer extension assay with the lagging-strand DNA polymerase delta. Surprisingly, however, FANCJ variants that are unable to bind an FeS cluster and to unwind DNA in vitro can partially suppress the formation of replisome-associated G4 structures that we observe in a FANCJ knock-out cell line. This may suggest a partially retained cellular activity of FANCJ variants with alterations in the FeS domain. On the other hand, FANCJ knock-out cells expressing FeS cluster-deficient variants display a similar-enhanced-sensitivity towards pyridostatin (PDS) and CX-5461, two agents that stabilise G4 structures, as FANCJ knock-out cells. Mutations in FANCJ that abolish FeS cluster binding may hence be predictive of an increased cellular sensitivity towards G4-stabilising agents.

The CIA Targeting Complex Is Highly Regulated and Provides Two Distinct Binding Sites for Client Iron-Sulfur Proteins.

The cytoplasmic iron-sulfur assembly (CIA) targeting complex is required for the transfer of an iron-sulfur (Fe-S) cluster to cytoplasmic and nuclear proteins, but how it engages with client proteins is unknown. Here, we show that the complex members MIP18 and CIAO1 associate with the C terminus of MMS19. By doing so, they form a docking site for Fe-S proteins that is disrupted in the absence of either MMS19 or MIP18. The Fe-S helicase XPD seems to be the only exception, since it can interact with MMS19 independently of MIP18 and CIAO1. We further show that the direct interaction between MMS19 and MIP18 is required to protect MIP18 from proteasomal degradation. Taken together, these data suggest a remarkably regulated interaction between the CIA targeting complex and client proteins and raise the possibility that Fe-S cluster transfer is controlled, at least in part, by the stability of the CIA targeting complex itself.