Members of the RecQ family of helicases are known for their roles in DNA repair, replication, and recombination. Mutations in the human RecQ helicases, WRN and BLM, cause Werner and Bloom syndromes, which are diseases characterized by genome instability and an increased risk of cancer. While WRN contains both a helicase and an exonuclease domain, the Drosophila melanogaster homolog, WRNexo, contains only the exonuclease domain. Therefore the Drosophila model system provides a unique opportunity to study the exonuclease functions of WRN separate from the helicase. We created a null allele of WRNexo via imprecise P-element excision. The null WRNexo mutants are not sensitive to double-strand break-inducing reagents, suggesting that the exonuclease does not play a key role in homologous recombination-mediated repair of DSBs. However, WRNexo mutant embryos have a reduced hatching frequency and larvae are sensitive to the replication fork-stalling reagent, hydroxyurea (HU), suggesting that WRNexo is important in responding to replication stress. The role of WRNexo in the HU-induced stress response is independent of Rad51. Interestingly, the hatching defect and HU sensitivity of WRNexo mutants do not occur in flies containing an exonuclease-dead copy of WRNexo, suggesting that the role of WRNexo in replication is independent of exonuclease activity. Additionally, WRNexo and Blm mutants exhibit similar sensitivity to HU and synthetic lethality in combination with mutations in structure-selective endonucleases. We propose that WRNexo and BLM interact to promote fork reversal following replication fork stalling and in their absence regressed forks are restarted through a Rad51-mediated process.
The cytosolic iron-sulfur cluster assembly (CIA) system biosynthesizes iron-sulfur (FeS) cluster cofactors for cytosolic and nuclear proteins. The yeast Cia2 protein is the central component of the targeting complex which identifies apo-protein targets in the final step of the pathway. Herein, we determine that Cia2 contains five conserved motifs distributed between an intrinsically disordered N-terminal domain and a C-terminal domain of unknown function 59 (DUF59). The disordered domain is dispensible for binding the other subunits of the targeting complex, Met18 and Cia1, and the apo-target Rad3 in vitro. While in vivo assays reveal that the C-terminal domain is sufficient to support viability, several phenotypic assays indicate that deletion of the N-terminal domain negatively impacts CIA function. We additionally establish that Glu208, located within a conserved motif found only in eukaryotic DUF59 proteins, is important for the Cia1-Cia2 interaction in vitro. In vivo, E208A-Cia2 results in a diminished activity of the cytosolic iron sulfur cluster protein, Leu1 but only modest effects on hydroxyurea or methylmethane sulfonate sensitivity. Finally, we demonstrate that neither of the two highly conserved motifs of the DUF59 domain are vital for any of Cia2's interactions in vitro yet mutation of the DPE motif in the DUF59 domain results in a nonfunctional allele in vivo. Our observation that four of the five highly conserved motifs of Cia2 are dispensable for targeting complex formation and apo-target binding suggests that Cia2 is not simply a protein-protein interaction mediator but it likely possesses an additional, currently cryptic, function during the final cluster insertion step of CIA.
The Cytosolic Iron Sulfur Cluster Assembly (CIA) pathway is a highly conserved pathway that assembles and inserts iron sulfur (FeS) cluster cofactors into a variety of target proteins. These targets are involved in many cellular processes including DNA repair, iron homeostasis, and nucleotide metabolism. For CIA targets to receive the FeS cluster, they must be recruited for FeS cluster insertion by the CIA targeting complex. Although it is known that the targeting complex recognizes and binds to targets, the mechanisms of target recognition are not fully understood. In this work, mutagenesis and affinity co‐purification were utilized to elucidate the binding interface of Met18, a protein in the CIA targeting complex, and Rad3, a DNA helicase that receives an FeS cluster from the CIA pathway. Although this interaction has been investigated by other groups, there has been contradiction as to which portion of Rad3 binds to the targeting complex. Vashist et al. (J. Biol. Chem., 2012, 287, 43351) reported that the N‐terminus of Rad3 is responsible for binding to the targeting complex, while Ito et al. (Mol. Cell., 2010, 39, 632) reported that the C‐terminus of Rad3 is responsible for binding. To help resolve this discrepancy and gain insight into the targeting mechanism of the CIA targeting complex, truncated versions of Rad3 were expressed and isolated, and it was found that the N‐terminal region of the protein is responsible for binding to the targeting complex. Our current work involves investigating a 10 amino acid N‐terminal region of Rad3 identified by Vashist et al. Their group identified this region as participating in binding to Met18. In this work, we split the region into multiple alanine scans to further narrow down the Rad3‐Met18 binding interface. The identification of a region involved in Rad3‐Met18 binding could help define a target recognition motif for the CIA targeting complex.Support or Funding InformationBU Undergraduate Research Opportunities Program; NIH R01 GM121673This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.