Interaction map of Arabidopsis Mediator complex expounding its topology.

Understanding of mechanistic details of Mediator functioning in plants is impeded as the knowledge of subunit organization and structure is lacking. In this study, an interaction map of Arabidopsis Mediator complex was analyzed to understand the arrangement of the subunits in the core part of the complex. Combining this interaction map with homology-based modeling, probable structural topology of core part of the Arabidopsis Mediator complex was deduced. Though the overall topology of the complex was similar to that of yeast, several differences were observed. Many interactions discovered in this study are not yet reported in other systems. AtMed14 and AtMed17 emerged as the key component providing important scaffold for the whole complex. AtMed6 and AtMed10 were found to be important for linking head with middle and middle with tail, respectively. Some Mediator subunits were found to form homodimers and some were found to possess transactivation property. Subcellular localization suggested that many of the Mediator subunits might have functions beyond the process of transcription. Overall, this study reveals role of individual subunits in the organization of the core complex, which can be an important resource for understanding the molecular mechanism of functioning of Mediator complex and its subunits in plants.

Human hnRNPA1 reorganizes telomere-bound Replication Protein A.

Human replication protein A (RPA) is a heterotrimeric ssDNA binding protein responsible for many aspects of cellular DNA metabolism. Dynamic interactions of the four RPA DNA binding domains (DBDs) with DNA control replacement of RPA by downstream proteins in various cellular metabolic pathways. RPA plays several important functions at telomeres where it binds to and melts telomeric G-quadruplexes, non-canonical DNA structures formed at the G-rich telomeric ssDNA overhangs. Here, we combine single-molecule total internal reflection fluorescence microscopy (smTIRFM) and mass photometry (MP) with biophysical and biochemical analyses to demonstrate that heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) specifically remodels RPA bound to telomeric ssDNA by dampening the RPA configurational dynamics and forming a ternary complex. Uniquely, among hnRNPA1 target RNAs, telomeric repeat-containing RNA (TERRA) is selectively capable of releasing hnRNPA1 from the RPA-telomeric DNA complex. We speculate that this telomere specific RPA-DNA-hnRNPA1 complex is an important structure in telomere protection. One Sentence Summary: At the single-stranded ends of human telomeres, the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) binds to and modulates conformational dynamics of the ssDNA binding protein RPA forming a ternary complex which is controlled by telomeric repeat-containing RNA (TERRA).

Therapeutic disruption of RAD52-ssDNA complexation via novel drug-like inhibitors.

RAD52 protein is a coveted target for anticancer drug discovery. Similar to poly-ADP-ribose polymerase (PARP) inhibitors, pharmacological inhibition of RAD52 is synthetically lethal with defects in genome caretakers BRCA1 and BRCA2 (∼25% of breast and ovarian cancers). Emerging structure activity relationships for RAD52 are complex, making it challenging to transform previously identified disruptors of the RAD52-ssDNA interaction into drug-like leads using traditional medicinal chemistry approaches. Using pharmacophoric informatics on the RAD52 complexation by epigallocatechin (EGC), and the Enamine in silico REAL database, we identified six distinct chemical scaffolds that occupy the same physical space on RAD52 as EGC. All six were RAD52 inhibitors (IC50 ∼23-1200 µM) with two of the compounds (Z56 and Z99) selectively killing BRCA-mutant cells and inhibiting cellular activities of RAD52 at micromolar inhibitor concentrations. While Z56 had no effect on the ssDNA-binding protein RPA and was toxic to BRCA-mutant cells only, Z99 inhibited both proteins and displayed toxicity towards BRCA-complemented cells. Optimization of the Z99 scaffold resulted in a set of more powerful and selective inhibitors (IC50 ∼1.3-8 µM), which were only toxic to BRCA-mutant cells. RAD52 complexation by Z56, Z99 and its more specific derivatives provide a roadmap for next generation of cancer therapeutics.

A moving target for drug discovery: Structure activity relationship and many genome (de)stabilizing functions of the RAD52 protein.

BRCA-ness phenotype, a signature of many breast and ovarian cancers, manifests as deficiency in homologous recombination, and as defects in protection and repair of damaged DNA replication forks. A dependence of such cancers on DNA repair factors less important for survival of BRCA-proficient cells, offers opportunities for development of novel chemotherapeutic interventions. The first drugs targeting BRCA-deficient cancers, poly-ADP-ribose polymerase (PARP) inhibitors have been approved for the treatment of advanced, chemotherapy resistant cancers in patients with BRCA1/2 germline mutations. Nine additional proteins that can be targeted to selectively kill BRCA-deficient cancer cells have been identified. Among them, a DNA repair protein RAD52 is an especially attractive target due to general tolerance of the RAD52 loss of function, and protective role of an inactivating mutation. Yet, the effective pharmacological inhibitors of RAD52 have not been forthcoming. In this review, we discuss advances in the state of our knowledge of the RAD52 structure, activities and cellular functions, with a specific focus on the features that make RAD52 an attractive, but difficult drug target.

Expression of AtMed15 of Arabidopsis in yeast causes flocculation and increases ethanol production in yeast culture.

Mediator, a multiprotein complex involved in transcription of class II genes, was first discovered in yeast and then characterized in many metazoans revealing a striking structural conservation of the complex. However, sequences of Mediator subunits are not well conserved raising a question on the functional conservation of these individual subunits. In this study, expression of Med15 of Arabidopsis (AtMed15) in gal11∆ yeast could not complement the function of ScGal11 in galactose metabolism and resistance against cycloheximide. Surprisingly, AtMed15 changed the morphology of the yeast cells. The cells adhered strongly on the surface of the agar media, and showed robust flocculation in the liquid media without affecting the growth. The AtMed15-induced adhesion and flocculation were observed in different carbon sources. Calcium-assisted cell wall-bound mannan-binding proteins were found to be involved in this flocculation, which was unaffected by wide fluctuation of pH or temperatures revealing its constitutive robust nature. Expression of few flocculation related Flo genes was up-regulated in these cells. Interestingly, there was significant increase in ethanol production by the yeast expressing AtMed15. Robust and constitutive flocculation and increased ethanol production by yeast cells harbouring AtMed15 indicate an opportunity of its important usage in biotechnology industries.