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Summary: Fungal pathogens such as Candida albicans pose a significant threat to human health with limited treatment options available. One strategy to expand the therapeutic target space is to identify genes important for pathogen growth in host-relevant environments. Here, we leverage a pooled functional genomic screening strategy to identify genes important for fitness of C. albicans in diverse conditions. We identify an essential gene with no known Saccharomyces cerevisiae homolog, C1_09670C, and demonstrate that it encodes subunit 3 of replication factor A (Rfa3). Furthermore, we apply computational analyses to identify functionally coherent gene clusters and predict gene function. Through this approach, we predict the cell-cycle-associated function of C3_06880W, a previously uncharacterized gene required for fitness specifically at elevated temperatures, and follow-up assays confirm that C3_06880W encodes Iml3, a component of the C. albicans kinetochore with roles in virulence in vivo. Overall, this work reveals insights into the vulnerabilities of C. albicans.

Summary: Activation of the Fanconi anemia (FA) pathway after treatment with mitomycin C (MMC) is essential for preventing chromosome translocations termed “radials.” When replication forks stall at MMC-induced interstrand crosslinks (ICLs), the FA pathway is activated to orchestrate ICL unhooking and repair of the DNA break intermediates. However, in FA-deficient cells, how ICL-associated breaks are resolved in a manner that leads to radials is unclear. Here, we demonstrate that MMC-induced radials are dependent on DNA polymerase theta (POLθ)-mediated alternative end joining (A-EJ). Specifically, we show that radials observed in FANCD2−/− cells are dependent on POLθ and DNA ligase III and occur independently of classical non-homologous end joining. Furthermore, treatment of FANCD2−/− cells with POLθ inhibitors abolishes radials and leads to the accumulation of breaks co-localizing with common fragile sites. Uniformly, these observations implicate A-EJ in radial formation and provide mechanistic insights into the treatment of FA pathway-deficient cancers with POLθ inhibitors.

A comprehensive view of molecular chaperone function in the cell was obtained through a systematic global integrative network approach based on physical (protein-protein) and genetic (gene-gene or epistatic) interaction mapping. This allowed us to decipher interactions involving all core chaperones (67) and cochaperones (15) of Saccharomyces cerevisiae. Our analysis revealed the presence of a large chaperone functional supercomplex, which we named the naturally joined (NAJ) chaperone complex, encompassing Hsp40, Hsp70, Hsp90, AAA+, CCT, and small Hsps. We further found that many chaperones interact with proteins that form foci or condensates under stress conditions. Using an in vitro reconstitution approach, we demonstrate condensate formation for the highly conserved AAA+ ATPases Rvb1 and Rvb2, which are part of the R2TP complex that interacts with Hsp90. This expanded view of the chaperone network in the cell clearly demonstrates the distinction between chaperones having broad versus narrow substrate specificities in protein homeostasis.

Summary: To systematically explore the genes mediating functional crosstalk between metazoan biological processes, we apply comparative genetic interaction (GI) mapping in Saccharomyces cerevisiae and Caenorhabditis elegans to generate an inter-bioprocess network consisting of 178 C. elegans GIs. The GI network spans six annotated biological processes including aging, intracellular transport, microtubule-based processes, cytokinesis, lipid metabolic processes, and anatomical structure development. By proposing a strategy called “reciprocal functional test” for interacting gene pairs, we discover a group of genes that mediate crosstalk between distinct biological processes. In particular, we identify the ribosomal S6 Kinase/RSKS-1, previously characterized as an mTOR (mechanistic target of rapamycin) effector, as a regulator of DAF-2 endosomal recycling transport, which traces a functional correlation between endocytic recycling and aging processes. Together, our results provide an alternative and effective strategy for identifying genes and pathways that mediate crosstalk between bioprocesses with little prior knowledge. : Chen et al. develop a “reciprocal functional test” to identify genes mediating crosstalk between different bioprocesses with little prior knowledge. With this method, ribosomal S6 Kinase/RSKS-1 is identified as a potential regulator of DAF-2 recycling. This finding traces a functional link between endocytic recycling and aging processes. Keywords: genetic interaction, bioprocess crosstalk, comparative mapping, endocytic recycling, RSKS-1/S6 Kinase, Caenorhabditis elegans, Saccharomyces cerevisiae, RAB-10/Rab10, recycling endosomes, mTOR