The origin recognition complex (ORC) marks chromosomal sites as replication origins and is essential for replication initiation. In yeast, ORC also binds to DNA elements called silencers, where its primary function is to recruit silent information regulator (SIR) proteins to establish transcriptional silencing. Indeed, silencers function poorly as chromosomal origins. Several genetic, molecular, and biochemical studies of HMR-E have led to a model proposing that when ORC becomes limiting in the cell (such as in the orc2-1 mutant) only sites that bind ORC tightly (such as HMR-E) remain fully occupied by ORC, while lower affinity sites, including many origins, lose ORC occupancy. Since HMR-E possessed a unique non-replication function, we reasoned that other tight sites might reveal novel functions for ORC on chromosomes. Therefore, we comprehensively determined ORC “affinity” genome-wide by performing an ORC ChIP–on–chip in ORC2 and orc2-1 strains. Here we describe a novel group of orc2-1–resistant ORC–interacting chromosomal sites (ORF–ORC sites) that did not function as replication origins or silencers. Instead, ORF–ORC sites were comprised of protein-coding regions of highly transcribed metabolic genes. In contrast to the ORC–silencer paradigm, transcriptional activation promoted ORC association with these genes. Remarkably, ORF–ORC genes were enriched in proximity to origins of replication and, in several instances, were transcriptionally regulated by these origins. Taken together, these results suggest a surprising connection among ORC, replication origins, and cellular metabolism.
The chromosome passenger complex (CPC) is an essential regulator of mitosis and cytokinesis. The CPC consists of Aurora B kinase, inner centromere protein (INCENP), and the targeting subunits survivin and borealin/Dasra B. INCENP is a scaffolding subunit for the CPC and activates Aurora B via its conserved IN-box domain. We show that overexpression of soluble IN-box in HeLa cells affects endogenous CPC localization and produces a significant increase in multinucleated and micronucleated cells consistent with CPC loss of function. The dominant-negative effect of soluble IN-box expression depends on residues corresponding to hINCENP W845 and/or F881, suggesting that these are essential for Aurora B binding in vivo. We then screened a targeted library of small (five to nine residues long) circular peptide (CP) IN-box fragments generated using split intein circular ligation of proteins and peptides (SICLOPPS) methodology. We identified a number of CPs that caused modest but reproducible increases in rates of multinucleated and micronucleated cells. Our results provide proof of concept that inhibition of the Aurora B–IN-box interaction is a viable strategy for interfering with CPC function in vivo.
Genetically-encoded cyclic peptide libraries allow rapid in vivo screens for inhibitors of any target protein of interest. In particular, the Split Intein Circular Ligation of Protein and Peptides (SICLOPPS) system exploits spontaneous protein splicing of inteins to produce intracellular cyclic peptides. A previous SICLOPPS screen against Aurora B kinase, which plays a critical role during chromosome segregation, identified several candidate inhibitors that we sought to recapitulate by chemical synthesis. We describe the syntheses of cyclic peptide hits and analogs via solution-phase macrocyclization of side chain-protected linear peptides obtained from standard solidphase peptide synthesis. Cyclic peptide targets, including cyclo-[CTWAR], were designed to match both the variable portions and conserved cysteine residue of their genetically-encoded counterparts. Synthetic products were characterized by tandem high-resolution mass spectrometry to analyze a combination of exact mass, isotopic pattern, and collisional dissociation-induced fragmentation pattern. The latter analyses facilitated the distinction between targets and oligomeric side products, and served to confirm peptidic sequences in a manner that can be readily extended to analyses of complex biological samples. This alternative chemical synthesis approach for cyclic peptides allows cost-effective validation and facile chemical elaboration of hit candidates from SICLOPPS screens. | INTRODUCTIONAccess to complex chemical matter is critical for the discovery of bioactive compounds that serve as chemical probes in biomedical research and as entry points for drug discovery. [1] Traditionally, either natural product extracts or synthetic chemistry have been used to generate compound libraries for high-throughput screens based on either in vitro biochemical assays or in vivo phenotypic readouts. New technologies promise to accelerate interrogation of bioactive chemical space including diversity-oriented synthesis, [2] DNA-encoded libraries, [3] combinatorial enzymatic biosynthesis of natural product-like compounds, [4,5] and artificial intelligence-based virtual screening. [6] Genetically-encoded peptide libraries, as produced in bacteriophage, bacteria, yeast, and in vitro formats, allow access to tremendous combinatorial diversity and have been successfully deployed against many important drug targets. [7] While linear peptides represent the simplest case for both genetic encoding and chemical synthesis, linear peptides suffer from several liabilities including entropic penalties during binding due to backbone flexibility, poor cellular uptake, rapid proteolytic degradation, and
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