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

Abstract

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. Breast and ovarian cancers are often linked to a genetic predisposition, most commonly through mutations in the breast cancer susceptibility genes BRCA1 and BRCA2, but also other genes, such as FANCJ/BRIP1, are associated with an increased disease risk. The small molecule CX-5461 is currently in phase I/II clinical trials for patients with BRCA-deficient tumours. It was originally identified as an rDNA transcription inhibitor, but–more recently–also found to bind and stabilise G4 DNA secondary structures. In this study we show that FANCJ-deficient cells have an increased sensitivity towards CX-5461. Our data further suggest that single amino acid changes in FANCJ that abolish its helicase activity or its ability to bind an iron-sulfur co-factor are sufficient to render cells more sensitive to CX-5461 treatment. Combined, our findings support a model whereby FANCJ resolves G4 structures in the context of the replisome to allow replication through guanine-rich regions of the genome. Mechanistically, FANCJ’s ability to resolve G4 structures largely depends on an intact helicase domain and partially on the iron-sulfur cluster-binding domain. The latter finding is important since a number of cancer-associated mutations are located within the iron-sulfur domain of FANCJ.