Faculty Bibliography

Transcription and replication both require large macromolecular complexes to act on a DNA template, yet these machineries cannot simultaneously act on the same DNA sequence. Conflicts between the replication and transcription machineries (transcription-replication conflicts, or TRCs) are widespread in both prokaryotes and eukaryotes and have the capacity to both cause DNA damage and compromise complete, faithful replication of the genome. This review will highlight recent studies investigating the genomic locations of TRCs and the mechanisms by which they may be prevented, mitigated, or resolved. We address work from both model organisms and mammalian systems but predominantly focus on multicellular eukaryotes owing to the additional complexities inherent in the coordination of replication and transcription in the context of cell type-specific gene expression and higher-order chromatin organization.

The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70-Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity.

Timely repair of chromosomal double-strand breaks is required for genome integrity and cellular viability. The polymerase theta-mediated end joining pathway has an important role in resolving these breaks and is essential in cancers defective in other DNA repair pathways, thus making it an emerging therapeutic target¹. It requires annealing of 2-6 nucleotides of complementary sequence, microhomologies, that are adjacent to the broken ends, followed by initiation of end-bridging DNA synthesis by polymerase θ. However, the other pathway steps remain inadequately defined, and the enzymes required for them are unknown. Here we demonstrate requirements for exonucleolytic digestion of unpaired 3' tails before polymerase θ can initiate synthesis, then a switch to a more accurate, processive and strand-displacing polymerase to complete repair. We show the replicative polymerase, polymerase δ, is required for both steps; its 3' to 5' exonuclease activity for flap trimming, then its polymerase activity for extension and completion of repair. The enzymatic steps that are essential and specific to this pathway are mediated by two separate, sequential engagements of the two polymerases. The requisite coupling of these steps together is likely to be facilitated by physical association of the two polymerases. This pairing of polymerase δ with a polymerase capable of end-bridging synthesis, polymerase θ, may help to explain why the normally high-fidelity polymerase δ participates in genome destabilizing processes such as mitotic DNA synthesis² and microhomology-mediated break-induced replication³.

Generation of virus-host protein-protein interactions (PPIs) maps may provide clues to uncover SARS-CoV-2-hijacked cellular processes. However, these PPIs maps were created by expressing each viral protein singularly, which does not reflect the life situation in which certain viral proteins synergistically interact with host proteins. Our results reveal the host-viral protein-protein interactome of SARS-CoV-2 NSP3, NSP4, and NSP6 expressed individually or in combination. Furthermore, REEP5/TRAM1 complex interacts with NSP3 at ROs and promotes viral replication. The significance of our research is identifying virus-host interactions that may be targeted for therapeutic intervention.

Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient-poor conditions, SKP1 is phosphorylated on Thr¹³¹, allowing its interaction with V₁ subunits of the vacuolar ATPase (V-ATPase). This event, in turn, promotes V-ATPase assembly to acidify late endosomes and enhance endolysosomal degradation. Under nutrient-rich conditions, SUMOylation of phosphorylated SKP1 allows its binding to and dephosphorylation by the PPM1B phosphatase. Dephosphorylated SKP1 interacts with SEC22B to promote unconventional secretion of the content of less acidified hybrid endosomal/autophagic compartments. Collectively, our study implicates SKP1 phosphorylation as a switch between autophagy and unconventional secretion in a manner dependent on cellular nutrient status.

Gene inactivation can affect the process(es) in which that gene acts and causally downstream ones, yielding diverse mutant phenotypes. Identifying the genetic pathways resulting in a given phenotype helps us understand how individual genes interact in a functional network. Computable representations of biological pathways include detailed process descriptions in the Reactome Knowledgebase and causal activity flows between molecular functions in Gene Ontology-Causal Activity Models (GO-CAMs). A computational process has been developed to convert Reactome pathways to GO-CAMs. Laboratory mice are widely used models of normal and pathological human processes. We have converted human Reactome GO-CAMs to orthologous mouse GO-CAMs, as a resource to transfer pathway knowledge between humans and model organisms. These mouse GO-CAMs allowed us to define sets of genes that function in a causally connected way. To demonstrate that individual variant genes from connected pathways result in similar but distinguishable phenotypes, we used the genes in our pathway models to cross-query mouse phenotype annotations in the Mouse Genome Database (MGD). Using GO-CAM representations of 2 related but distinct pathways, gluconeogenesis and glycolysis, we show that individual causal paths in gene networks give rise to discrete phenotypic outcomes resulting from perturbations of glycolytic and gluconeogenic genes. The accurate and detailed descriptions of gene interactions recovered in this analysis of well-studied processes suggest that this strategy can be applied to less well-understood processes in less well-studied model systems to predict phenotypic outcomes of novel gene variants and to identify potential gene targets in altered processes.

Prostate cancers adapt to androgen receptor (AR) pathway inhibitors and progress to castration resistance due to ongoing AR expression and function. To counter this, we developed a new approach to modulate the AR and inhibit castration-resistant prostate cancer (CRPC) using multivalent peptoid conjugates (MPC) that contain multiple copies of the AR-targeting ligand ethisterone attached to a peptidomimetic scaffold. Here, we investigated the antitumor effects of compound MPC309, a trivalent display of ethisterone conjugated to a peptoid oligomer backbone that binds to the AR with nanomolar affinity. MPC309 exhibited potent antiproliferative effects on various enzalutamide-resistant prostate cancer models, including those with AR splice variants, ligand-binding mutations, and noncanonical AR gene expression programs, as well as mouse prostate organoids harboring defined genetic alterations that mimic lethal human prostate cancer subtypes. MPC309 is taken up by cells through macropinocytosis, an endocytic process more prevalent in cancer cells than in normal ones, thus providing an opportunity to target tumors selectively. MPC309 triggers a distinct AR transcriptome compared with DHT and enzalutamide, a clinically used antiandrogen. Specifically, MPC309 enhances the expression of differentiation genes while reducing the expression of genes needed for cell division and metabolism. Mechanistically, MPC309 increases AR chromatin occupancy and alters AR interactions with coregulatory proteins in a pattern distinct from DHT. In xenograft studies, MPC309 produced significantly greater tumor suppression than enzalutamide. Altogether, MPC309 represents a promising new AR modulator that can combat resistant disease by promoting an AR antiproliferative gene expression program.

Antibiotic binding sites are located in important domains of essential enzymes and have been extensively studied in the context of resistance mutations; however, their study is limited by positive selection. Using multiplex genome engineering¹ to overcome this constraint, we generate and characterize a collection of 760 single-residue mutants encompassing the entire rifampicin binding site of Escherichia coli RNA polymerase (RNAP). By genetically mapping drug-enzyme interactions, we identify an alpha helix where mutations considerably enhance or disrupt rifampicin binding. We find mutations in this region that prolong antibiotic binding, converting rifampicin from a bacteriostatic to bactericidal drug by inducing lethal DNA breaks. The latter are replication dependent, indicating that rifampicin kills by causing detrimental transcription-replication conflicts at promoters. We also identify additional binding site mutations that greatly increase the speed of RNAP.Fast RNAP depletes the cell of nucleotides, alters cell sensitivity to different antibiotics and provides a cold growth advantage. Finally, by mapping natural rpoB sequence diversity, we discover that functional rifampicin binding site mutations that alter RNAP properties or confer drug resistance occur frequently in nature.