RESUMEN
The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.
Asunto(s)
Antineoplásicos , Descubrimiento de Drogas , Inhibidores Enzimáticos , Inestabilidad de Microsatélites , Neoplasias , Mutaciones Letales Sintéticas , Helicasa del Síndrome de Werner , Animales , Femenino , Humanos , Ratones , Administración Oral , Regulación Alostérica/efectos de los fármacos , Antineoplásicos/efectos adversos , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Ensayos Clínicos como Asunto , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Daño del ADN/efectos de los fármacos , Inhibidores Enzimáticos/efectos adversos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Neoplasias/metabolismo , Dominios Proteicos , Reproducibilidad de los Resultados , Supresión Genética , Mutaciones Letales Sintéticas/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Helicasa del Síndrome de Werner/antagonistas & inhibidores , Helicasa del Síndrome de Werner/genética , Helicasa del Síndrome de Werner/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RNase P is an essential enzyme found across all domains of life that is responsible for the 5'-end maturation of precursor tRNAs. For decades, numerous studies have sought to elucidate the mechanisms and biochemistry governing RNase P function. However, much remains unknown about the regulation of RNase P expression, the turnover and degradation of the enzyme, and the mechanisms underlying the phenotypes and complementation of specific RNase P mutations, especially in the model bacterium, Escherichia coli In E. coli, the temperature-sensitive (ts) rnpA49 mutation in the protein subunit of RNase P has arguably been one of the most well-studied mutations for examining the enzyme's activity in vivo. Here, we report for the first time naturally occurring temperature-resistant suppressor mutations of E. coli strains carrying the rnpA49 allele. We find that rnpA49 strains can partially compensate the ts defect via gene amplifications of either RNase P subunit (rnpA49 or rnpB) or by the acquisition of loss-of-function mutations in Lon protease or RNase R. Our results agree with previous plasmid overexpression and gene deletion complementation studies, and importantly suggest the involvement of Lon protease in the degradation and/or regulatory pathway(s) of the mutant protein subunit of RNase P. This work offers novel insights into the behavior and complementation of the rnpA49 allele in vivo and provides direction for follow-up studies regarding RNase P regulation and turnover in E. coli.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Mutación , Fenotipo , Ribonucleasa P , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ribonucleasa P/genética , Ribonucleasa P/metabolismo , Proteasa La/genética , Proteasa La/metabolismo , Supresión Genética , TemperaturaRESUMEN
Nuclear egress is an essential process in herpesvirus replication whereby nascent capsids translocate from the nucleus to the cytoplasm. This initial step of nuclear egress-budding at the inner nuclear membrane-is coordinated by the nuclear egress complex (NEC). Composed of the viral proteins UL31 and UL34, NEC deforms the membrane around the capsid as the latter buds into the perinuclear space. NEC oligomerization into a hexagonal membrane-bound lattice is essential for budding because NEC mutants designed to perturb lattice interfaces reduce its budding ability. Previously, we identified an NEC suppressor mutation capable of restoring budding to a mutant with a weakened hexagonal lattice. Using an established in-vitro budding assay and HSV-1 infected cell experiments, we show that the suppressor mutation can restore budding to a broad range of budding-deficient NEC mutants thereby acting as a universal suppressor. Cryogenic electron tomography of the suppressor NEC mutant lattice revealed a hexagonal lattice reminiscent of wild-type NEC lattice instead of an alternative lattice. Further investigation using x-ray crystallography showed that the suppressor mutation promoted the formation of new contacts between the NEC hexamers that, ostensibly, stabilized the hexagonal lattice. This stabilization strategy is powerful enough to override the otherwise deleterious effects of mutations that destabilize the NEC lattice by different mechanisms, resulting in a functional NEC hexagonal lattice and restoration of membrane budding.
Asunto(s)
Herpesviridae , Herpesvirus Humano 1 , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Supresión Genética , Núcleo Celular/metabolismo , Membrana Nuclear/metabolismo , Herpesviridae/metabolismo , Liberación del VirusRESUMEN
Targeted gene regulation is indispensable for exploring gene functions in microbes and the development of microbial cell factories. While most loci can be regulated by CRISPRi, it cannot be used for targets lacking protospacer adjacent motifs (PAM) or protospacer flanking sequences (PFS). Here, we characterized a genetic suppression approach named the hpDNA-assisted structure-guided nuclease mediating interference system (HpSGNi). It was composed of a flap endonuclease 1 (FEN1) and mis-hairpin DNA probes (mis-hpDNA) to suppress the expression of target genes simply and efficiently in microbe without sequence restrictions. By inhibiting the initiation and elongation of the transcription, HpSGNi successfully silenced the transcription of exogenous fluorescent protein genes, ampicillin resistance gene and endogenous folP/sulA genes in Escherichia coli BL21(DE3) and K-12 MG1655. Meanwhile, aiming at optimizing the mis-hpDNA, we displayed the characteristics by detecting the tolerance to the single base mismatch and length of the guide sequence. This DNA-guided recognition platform provides a simple approach for selectively inhibiting gene expression.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Supresión Genética , ADN , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Sondas de ADN/metabolismoRESUMEN
BACKGROUND: Genetic suppression occurs when the deleterious effects of a primary "query" mutation, such as a disease-causing mutation, are rescued by a suppressor mutation elsewhere in the genome. METHODS: To capture existing knowledge on suppression relationships between human genes, we examined 2,400 published papers for potential interactions identified through either genetic modification of cultured human cells or through association studies in patients. RESULTS: The resulting network encompassed 476 unique suppression interactions covering a wide spectrum of diseases and biological functions. The interactions frequently linked genes that operate in the same biological process. Suppressors were strongly enriched for genes with a role in stress response or signaling, suggesting that deleterious mutations can often be buffered by modulating signaling cascades or immune responses. Suppressor mutations tended to be deleterious when they occurred in absence of the query mutation, in apparent contrast with their protective role in the presence of the query. We formulated and quantified mechanisms of genetic suppression that could explain 71% of interactions and provided mechanistic insight into disease pathology. Finally, we used these observations to predict suppressor genes in the human genome. CONCLUSIONS: The global suppression network allowed us to define principles of genetic suppression that were conserved across diseases, model systems, and species. The emerging frequency of suppression interactions among human genes and range of underlying mechanisms, together with the prevalence of suppression in model organisms, suggest that compensatory mutations may exist for most genetic diseases.
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Genoma Humano , Supresión Genética , Humanos , Mutación , Modelos Biológicos , Genética HumanaRESUMEN
Human P-glycoprotein (P-gp) or ABCB1 is overexpressed in many cancers and has been implicated in altering the bioavailability of chemotherapeutic drugs due to their efflux, resulting in the development of chemoresistance. To elucidate the mechanistic aspects and structure-function relationships of P-gp, we previously utilized a tyrosine (Y)-enriched P-gp mutant (15Y) and demonstrated that at least 15 conserved residues in the drug-binding pocket of P-gp are responsible for optimal substrate interaction and transport. To further understand the role of these 15 residues, two new mutants were generated, namely 6Y with the substitution of six residues (F72, F303, I306, F314, F336 and L339) with Y in transmembrane domain (TMD) 1 and 9Y with nine substitutions (F732, F759, F770, F938, F942, M949, L975, F983 and F994) in TMD2. Although both the mutants were expressed at normal levels at the cell surface, the 6Y mutant failed to transport all the tested substrates except Bodipy-verapamil, whereas the 9Y mutant effluxed all tested substrates in a manner very similar to that of the wild-type protein. Further mutational analysis revealed that two second-site mutations, one in intracellular helix (ICH) 4 (F916Y) and one in the Q loop of nucleotide-binding domain (NBD) 1 (F480Y) restored the transport function of 6Y. Additional biochemical data and comparative molecular dynamics simulations of the 6Y and 6Y+F916Y mutant indicate that the Q-loop of NBD1 of P-gp communicates with the substrate-binding sites in the transmembrane region through ICH4. This is the first evidence for the existence of second-site suppressors in human P-gp that allow recovery of the loss of transport function caused by primary mutations. Further study of such mutations could facilitate mapping of the communication pathway between the substrate-binding pocket and the NBDs of P-gp and possibly other ABC drug transporters.
Asunto(s)
Neoplasias , Supresión Genética , Humanos , Mutación , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP , NucleótidosRESUMEN
By targeting mRNA transcripts, non-coding small RNAs (sRNAs) regulate the expression of genes governing a wide range of bacterial functions. In the social myxobacterium Myxococcus xanthus, the sRNA Pxr serves as a gatekeeper of the regulatory pathway controlling the life-cycle transition from vegetative growth to multicellular fruiting body development. When nutrients are abundant, Pxr prevents the initiation of the developmental program, but Pxr-mediated inhibition is alleviated when cells starve. To identify genes essential for Pxr function, a developmentally defective strain in which Pxr-mediated blockage of development is constitutively active (strain "OC") was transposon-mutagenized to identify suppressor mutations that inactivate or bypass Pxr inhibition and thereby restore development. One of the four loci in which a transposon insertion restored development is rnd, encoding the Ribonuclease D protein (RNase D). RNase D is an exonuclease important for tRNA maturation. Here, we show that disruption of rnd abolishes the accumulation of Pxr-S, the product of Pxr processing from a longer precursor form (Pxr-L) and the active inhibitor of development. Additionally, the decrease in Pxr-S caused by rnd disruption was associated with increased accumulation primarily of a longer novel Pxr-specific transcript (Pxr-XL) rather than of Pxr-L. The introduction of a plasmid expressing rnd reverted cells back to OC-like phenotypes in development and Pxr accumulation, indicating that a lack of RNase D alone suppresses the developmental defect of OC. Moreover, an in vitro Pxr-processing assay demonstrated that RNase D processes Pxr-XL into Pxr-L; this implies that overall, Pxr sRNA maturation requires a sequential two-step processing. Collectively, our results indicate that a housekeeping ribonuclease plays a central role in a model form of microbial aggregative development. To our knowledge, this is the first evidence implicating RNase D in sRNA processing.
Asunto(s)
Myxococcales , ARN Pequeño no Traducido , Ribonucleasa III/genética , ARN Bacteriano/genética , Myxococcales/genética , Supresión Genética , ARN Pequeño no Traducido/genéticaRESUMEN
Nonsense mutations trigger premature translation termination and often give rise to prevalent and rare genetic diseases. Consequently, the pharmacological suppression of an unscheduled stop codon represents an attractive treatment option and is of high clinical relevance. At the molecular level, the ability of the ribosome to continue translation past a stop codon is designated stop codon readthrough (SCR). SCR of disease-causing premature termination codons (PTCs) is minimal but small molecule interventions, such as treatment with aminoglycoside antibiotics, can enhance its frequency. In this review, we summarize the current understanding of translation termination (both at PTCs and at cognate stop codons) and highlight recently discovered pathways that influence its fidelity. We describe the mechanisms involved in the recognition and readthrough of PTCs and report on SCR-inducing compounds currently explored in preclinical research and clinical trials. We conclude by reviewing the ongoing attempts of personalized nonsense suppression therapy in different disease contexts, including the genetic skin condition epidermolysis bullosa.
Asunto(s)
Codón sin Sentido , Enfermedades Genéticas Congénitas , Extensión de la Cadena Peptídica de Translación , Medicina de Precisión , Enfermedades Raras , Supresión Genética , Animales , Humanos , Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Codón sin Sentido/genética , Fibrosis Quística/genética , Fibrosis Quística/terapia , Epidermólisis Ampollosa/genética , Epidermólisis Ampollosa/terapia , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/terapia , Nefritis Hereditaria/genética , Nefritis Hereditaria/terapia , Degradación de ARNm Mediada por Codón sin Sentido , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Medicina de Precisión/métodos , Medicina de Precisión/tendencias , Enfermedades Raras/genética , Enfermedades Raras/terapia , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/terapia , Síndrome de Shwachman-Diamond/genética , Síndrome de Shwachman-Diamond/terapia , Supresión Genética/efectos de los fármacos , Supresión Genética/genética , Terminación de la Cadena Péptídica Traduccional/efectos de los fármacos , Aminoglicósidos/farmacologíaRESUMEN
Polo-like kinase 1 (Plk1) is a mitotic kinase that has multiple functions throughout the cell cycle. Catalytic activation of Plk1 is known to be regulated by phosphorylation of the kinase domain, including Thr210, and by releasing the kinase domain from its inhibitory polo-box domain. However, how Plk1 is activated to fulfill its proper roles, in time and space, is not well understood. In this study, we unintentionally found that the expression of a constitutively active form of human Plk1 is toxic to bacterial cells, such that cells contained point mutations that alleviate the kinase activity. Structural prediction revealed that these mutations are adjacent to the amino acids supporting the kinase activity. When human cells express these mutants, we found decreased levels of Plk1's substrate phosphorylation, resulting in mitotic defects. Moreover, unlike in bacterial cells, the expression of activated Plk1 mutants did not affect cell proliferation in human cells unless localized at the right place in mitosis. Our observations identified new suppressor mutations and underscored the importance of spatiotemporal regulation in Plk1, providing a basis for how we might intervene in this kinase for therapeutic purpose in human cells.
Asunto(s)
Proteínas de Ciclo Celular , Supresión Genética , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Mitosis/genética , Quinasa Tipo Polo 1RESUMEN
Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) of many Gram-negative bacteria, providing a barrier against the entry of toxic molecules. In Escherichia coli, LPS is exported to the cell surface by seven essential proteins (LptA-G) that form a transenvelope complex. At the inner membrane, the ATP-binding cassette (ABC) transporter LptB2FG associates with LptC to power LPS extraction from the membrane and transfer to the periplasmic LptA protein, which is in complex with the OM translocon LptDE. LptC interacts both with LptB2FG and LptADE to mediate the formation of the transenvelope bridge and regulates the ATPase activity of LptB2FG. A genetic screen has previously identified suppressor mutants at a residue (R212) of LptF that are viable in the absence of LptC. Here, we present in vivo evidence that the LptF R212G mutant assembles a six-protein transenvelope complex in which LptA mediates interactions with LptF and LptD in the absence of LptC. Furthermore, we present in vitro evidence that the mutant LptB2FG complexes restore the regulation of ATP hydrolysis as it occurs in the LptB2FGC complex to achieve wild-type efficient coupling of ATP hydrolysis and LPS movement. We also show the suppressor mutations restore the wild-type levels of LPS transport both in vivo and in vitro, but remarkably, without restoring the affinity of the inner membrane complex for LptA. Based on the sensitivity of lptF suppressor mutants to selected stress conditions relative to wild-type cells, we show that there are additional regulatory functions of LptF and LptC that had not been identified. IMPORTANCE The presence of an external LPS layer in the outer membrane makes Gram-negative bacteria intrinsically resistant to many antibiotics. Millions of LPS molecules are transported to the cell surface per generation by the Lpt molecular machine made, in E. coli, by seven essential proteins. LptC is the unconventional regulatory subunit of the LptB2FGC ABC transporter, involved in coordinating energy production and LPS transport. Surprisingly, despite being essential for bacterial growth, LptC can be deleted, provided that a specific residue in the periplasmic domain of LptF is mutated and LptA is overexpressed. Here, we apply biochemical techniques to investigate the suppression mechanism. The data produced in this work disclose an unknown regulatory function of LptF in the transporter that not only expands the knowledge about the Lpt complex but can also be targeted by novel LPS biogenesis inhibitors.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Lipopolisacáridos/metabolismo , Supresión Genética , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Transporte Biológico/fisiología , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Portadoras/metabolismoRESUMEN
Biosyntheses of para-aminobenzoic acid (PABA) and its downstream folic acid metabolites are essential for one-carbon metabolism in all life forms and the targets of sulfonamide and trimethoprim antibiotics. In this study, we identified and characterized two genes (pabA and pabBC) required for PABA biosynthesis in Listeria monocytogenes. Mutants in PABA biosynthesis were able to grow normally in rich media but not in defined media lacking PABA, but growth was restored by the addition of PABA or its downstream metabolites. PABA biosynthesis mutants were attenuated for intracellular growth in bone marrow-derived macrophages, produced extremely small plaques in fibroblast monolayers, and were highly attenuated for virulence in mice. PABA biosynthesis genes were upregulated upon infection and induced during growth in broth in a strain in which the master virulence regulator, PrfA, was genetically locked in its active state (PrfA*). To gain further insight into why PABA mutants were so attenuated, we screened for transposon-induced suppressor mutations that formed larger plaques. Suppressor mutants in relA, which are predicted to have higher levels of (p)ppGpp, and mutants in codY, which is a GTP-binding repressor of many biosynthetic genes, partially rescued the plaque defect but, notably, restored the capacity of the mutants to escape from phagosomes and induce the polymerization of host cell actin. However, these suppressor mutant strains remained attenuated for virulence in mice. These data suggest that even though folic acid metabolites exist in host cells and might be available during infection, de novo synthesis of PABA is required for L. monocytogenes pathogenesis.
Asunto(s)
Listeria monocytogenes , Ratones , Animales , Ácido 4-Aminobenzoico/metabolismo , Virulencia/genética , Supresión Genética , Ácido Fólico/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión GénicaRESUMEN
Investigations on gene essentiality have important implications in several fields of basic and applied research. A variety of strategies have been developed over the years to identify essential genes. Here, we describe an implemented plasmid shuffling method useful to assess the essentiality of overlapped genes under very stringent conditions. A host strain harboring the chromosomal deletion of the genes of interest is complemented by a thermosensitive plasmid carrying the copy of gene 1, gene 2, and rpsL allele, conferring streptomycin sensitivity to an otherwise resistant strain. A compatible plasmid harboring a different selectable marker and the copy of gene 2 only is transformed into the host strain, resulting in the coexistence of two plasmids. These cells are grown at high temperatures in a medium containing streptomycin. Under such conditions, viable cells are expected to contain only the incoming plasmid and to carry suppressor mutation(s) that bypass the loss of the essential gene 1. The system may thus represent a valuable tool to identify interactions between essential proteins and cell pathways.
Asunto(s)
Genes Esenciales , Estreptomicina , Alelos , Mutación , Plásmidos/genética , Estreptomicina/farmacología , Supresión GenéticaRESUMEN
Lipopolysaccharide (LPS) constitutes the major component of the outer membrane and is essential for bacteria, such as Escherichia coli. Recent work has revealed the essential roles of LapB and LapC proteins in regulating LPS amounts; although, if any additional partners are involved is unknown. Examination of proteins co-purifying with LapB identified LapD as a new partner. The purification of LapD reveals that it forms a complex with several proteins involved in LPS and phospholipid biosynthesis, including FtsH-LapA/B and Fab enzymes. Loss of LapD causes a reduction in LpxC amounts and vancomycin sensitivity, which can be restored by mutations that stabilize LpxC (mutations in lapB, ftsH and lpxC genes), revealing that LapD acts upstream of LapB-FtsH in regulating LpxC amounts. Interestingly, LapD absence results in the substantial retention of LPS in the inner membranes and synthetic lethality when either the lauroyl or the myristoyl acyl transferase is absent, which can be overcome by single-amino acid suppressor mutations in LPS flippase MsbA, suggesting LPS translocation defects in ΔlapD bacteria. Several genes whose products are involved in cell envelope homeostasis, including clsA, waaC, tig and micA, become essential in LapD's absence. Furthermore, the overproduction of acyl carrier protein AcpP or transcriptional factors DksA, SrrA can overcome certain defects of the LapD-lacking strain.
Asunto(s)
Amidohidrolasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Lipopolisacáridos , Oxidorreductasas/metabolismo , Aciltransferasas/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Lipopolisacáridos/metabolismo , Supresión GenéticaRESUMEN
Spn1/Iws1 is an essential eukaryotic transcription elongation factor that is conserved from yeast to humans as an integral member of the RNA polymerase II elongation complex. Several studies have shown that Spn1 functions as a histone chaperone to control transcription, RNA splicing, genome stability, and histone modifications. However, the precise role of Spn1 is not understood, and there is little understanding of why it is essential for viability. To address these issues, we have isolated 8 suppressor mutations that bypass the essential requirement for Spn1 in Saccharomyces cerevisiae. Unexpectedly, the suppressors identify several functionally distinct complexes and activities, including the histone chaperone FACT, the histone methyltransferase Set2, the Rpd3S histone deacetylase complex, the histone acetyltransferase Rtt109, the nucleosome remodeler Chd1, and a member of the SAGA coactivator complex, Sgf73. The identification of these distinct groups suggests that there are multiple ways in which Spn1 bypass can occur, including changes in histone acetylation and alterations in other histone chaperones. Thus, Spn1 may function to overcome repressive chromatin by multiple mechanisms during transcription. Our results suggest that bypassing a subset of these functions allows viability in the absence of Spn1.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Factores de Elongación Transcripcional , Cromatina , Proteínas de Unión al ADN/genética , Histona Acetiltransferasas/genética , Chaperonas de Histonas/genética , Histona Desacetilasas/genética , Histona Metiltransferasas/genética , Histonas/genética , Nucleosomas , Factores de Elongación de Péptidos/genética , ARN Polimerasa II/genética , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Supresión Genética , Factores de Transcripción/genética , Transcripción Genética , Factores de Elongación Transcripcional/genéticaRESUMEN
Most amino acid substitutions in a protein either lead to partial loss-of-function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss-of-function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examined six global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 ß-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. When coupled to inactive mutants, they promote increased in-vivo solubilities as well as regain-of-function phenotypes. In the case of CcdB, where novel suppressors were isolated, we determined the crystal structures of three such suppressors to obtain insight into the specific molecular interactions responsible for the observed effects. While most individual suppressors result in small stability enhancements relative to wildtype, which can be combined to yield significant stability increments, thermodynamic stabilisation is neither necessary nor sufficient for suppressor action. Instead, in diverse systems, we observe that individual global suppressors greatly enhance the foldability of buried site mutants, primarily through increase in refolding rate parameters measured in vitro. In the crowded intracellular environment, mutations that slow down folding likely facilitate off-pathway aggregation. We suggest that suppressor mutations that accelerate refolding can counteract this, enhancing the yield of properly folded, functional protein in vivo.
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COVID-19 , SARS-CoV-2 , Humanos , Mutación , Pliegue de Proteína , Proteínas , Supresión GenéticaRESUMEN
Bacterial RNA polymerase (RNAP) coordinates transcription with DNA repair and replication. Many RNAP mutations have pleiotropic phenotypes with profound effects on transcription-coupled processes. One class of RNAP mutations (rpo∗) has been shown to suppress mutations in regulatory factors responsible for changes in gene expression during stationary phase or starvation, as well as in factors involved in the restoration of replication forks after DNA damage. These mutations were suggested to affect the ability of RNAP to transcribe damaged DNA and to decrease the stability of transcription complexes, thus facilitating their dislodging during DNA replication and repair, although this was not explicitly demonstrated. Here, we obtained nine mutations of this class located around the DNA/RNA binding cleft of Escherichia coli RNAP and analyzed their transcription properties in vitro. We found that these mutations decreased promoter complex stability to varying degrees, and all decreased the activity of rRNA promoters. However, they did not have strong effects on elongation complex stability. Some mutations were shown to stimulate transcriptional pauses or decrease intrinsic RNA cleavage by RNAP, but none altered the ability of RNAP to transcribe DNA templates containing damaged nucleotides. Thus, we conclude that the suppressor phenotypes of the mutations are unlikely to result from direct effects on DNA lesion recognition by RNAP but may be primarily explained by changes in transcription initiation. Further analysis of the effects of these mutations on the genomic distribution of RNAP and its interactions with regulatory factors will be essential for understanding their diverse phenotypes in vivo.
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ARN Polimerasas Dirigidas por ADN , Proteínas de Escherichia coli , Escherichia coli , Supresión Genética , Reparación del ADN , Replicación del ADN , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/genética , Transcripción GenéticaRESUMEN
Thermodynamic stability represents one important constraint on protein evolution, but the molecular basis for how mutations that change stability impact fitness remains unclear. Here, we demonstrate that a prevalent global suppressor mutation in TEM ß-lactamase, M182T, increases fitness by reducing proteolysis in vivo. We also show that a synthetic mutation, M182S, can act as a global suppressor and suggest that its absence from natural populations is due to genetic inaccessibility rather than fundamental differences in the protein's stability or activity.
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Escherichia coli , Supresión Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Evolución Molecular , Mutación , Termodinámica , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
Despite the thousands of spore germinant receptor operons identified in genomes of bacilli and clostridia, understanding how the three essential receptor components act as a signal transduction machine in germination remains limited. The paper by Amon et al. in this issue uses the classical genetic approach of suppression to define a region of likely interaction between the GerAA and GerAB proteins: it provides a first glimpse into potential events within the receptor complex (J. D. Amon, L. Artzi, and D. Z. Rudner, J Bacteriol 204:e00470-21, 2022, https://doi.org/10.1128/JB.00470-21).
Asunto(s)
Bacillus subtilis , Esporas Bacterianas , Bacillus subtilis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de la Membrana/genética , Esporas Bacterianas/genética , Supresión GenéticaRESUMEN
For decades, biologist have exploited the near boundless advantages that molecular and genetic tools and analysis provide for our ability to understand biological systems. One of these genetic tools, suppressor analysis, has proven invaluable in furthering our understanding of biological processes and pathways and in discovering unknown interactions between genes and gene products. The power of suppressor analysis lies in its ability to discover genetic interactions in an unbiased manner, often leading to surprising discoveries. With advancements in technology, high-throughput approaches have aided in large-scale identification of suppressors and have helped provide insight into the core functional mechanisms through which suppressors act. In this review, we examine some of the fundamental discoveries that have been made possible through analysis of suppressor mutations. In addition, we cover the different types of suppressor mutants that can be isolated and the biological insights afforded by each type. Moreover, we provide considerations for the design of experiments to isolate suppressor mutants and for strategies to identify intergenic suppressor mutations. Finally, we provide guidance and example protocols for the isolation and mapping of suppressor mutants.
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Epistasis Genética , Supresión GenéticaRESUMEN
Uncoordinated clashes between replication forks and transcription cause replication stress and genome instability, which are hallmarks of cancer and neurodegeneration. Here, we investigate the outcomes of head-on replication-transcription collisions, using as a model system budding yeast mutants for the helicase Sen1, the ortholog of human Senataxin. We found that RNA Polymerase II accumulates together with RNA:DNA hybrids at sites of head-on collisions. The replication fork and RNA Polymerase II are both arrested during the clash, leading to DNA damage and, in the long run, the inhibition of gene expression. The inactivation of RNA Polymerase II elongation factors, such as the HMG-like protein Spt2 and the DISF and PAF complexes, but not alterations in chromatin structure, allows replication fork progression through transcribed regions. Attenuation of RNA Polymerase II elongation rescues RNA:DNA hybrid accumulation and DNA damage sensitivity caused by the absence of Sen1, but not of RNase H proteins, suggesting that such enzymes counteract toxic RNA:DNA hybrids at different stages of the cell cycle with Sen1 mainly acting in replication. We suggest that the main obstacle to replication fork progression is the elongating RNA Polymerase II engaged in an R-loop, rather than RNA:DNA hybrids per se or hybrid-associated chromatin modifications.