Добірка наукової літератури з теми "Genome caretakers"

ABSTRACT The mobility of the Ty1 retrotransposon in the yeast Saccharomyces cerevisiae is restricted by a large collection of proteins that preserve the integrity of the genome during replication. Several of these repressors of Ty1 transposition (Rtt)/genome caretakers are orthologs of mammalian retroviral restriction factors. In rtt/genome caretaker mutants, levels of Ty1 cDNA and mobility are increased; however, the mechanisms underlying Ty1 hypermobility in most rtt mutants are poorly characterized. Here, we show that either or both of two S-phase checkpoint pathways, the replication stress pathway and the DNA damage pathway, partially or strongly stimulate Ty1 mobility in 19 rtt/genome caretaker mutants. In contrast, neither checkpoint pathway is required for Ty1 hypermobility in two rtt mutants that are competent for genome maintenance. In rtt101Δ mutants, hypermobility is stimulated through the DNA damage pathway components Rad9, Rad24, Mec1, Rad53, and Dun1 but not Chk1. We provide evidence that Ty1 cDNA is not the direct target of the DNA damage pathway in rtt101Δ mutants; instead, levels of Ty1 integrase and reverse transcriptase proteins, as well as reverse transcriptase activity, are significantly elevated. We propose that DNA lesions created in the absence of Rtt/genome caretakers trigger S-phase checkpoint pathways to stimulate Ty1 reverse transcriptase activity.

To date, no evidence supports the fact that animals play a role in the epidemiology of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus infectious disease 2019 (COVID-19). However, several animal species are naturally susceptible to SARS-CoV-2 infection. Besides pets (cats, dogs, Syrian hamsters, and ferrets) and farm animals (minks), different zoo animal species have tested positive for SARS-CoV-2 (large felids and non-human primates). After the summer of 2020, a second wave of SARS-CoV-2 infection occurred in Barcelona (Spain), reaching a peak of positive cases in November. During that period, four lions (Panthera leo) at the Barcelona Zoo and three caretakers developed respiratory signs and tested positive for the SARS-CoV-2 antigen. Lion infection was monitored for several weeks and nasal, fecal, saliva, and blood samples were taken at different time-points. SARS-CoV-2 RNA was detected in nasal samples from all studied lions and the viral RNA was detected up to two weeks after the initial viral positive test in three out of four animals. The SARS-CoV-2 genome was also detected in the feces of animals at different times. Virus isolation was successful only from respiratory samples of two lions at an early time-point. The four animals developed neutralizing antibodies after the infection that were detectable four months after the initial diagnosis. The partial SARS-CoV-2 genome sequence from one animal caretaker was identical to the sequences obtained from lions. Chronology of the events, the viral dynamics, and the genomic data support human-to-lion transmission as the origin of infection.

Coronavirus disease (COVID-19) is an emerging infectious disease caused by SARS-CoV-2. Given the emergence of SARS-CoV-2 variants, continuous surveillance of SARS-CoV-2 in animals is important. To monitor SARS-CoV-2 infection in wildlife in Thailand, we collected 62 blood samples and nine nasal- and rectal-swab samples from captive tigers (Panthera tigris) in Ratchaburi province in Thailand during 2020–2021. A plaque reduction neutralization test (PRNT) was employed to detect SARS-CoV-2 neutralizing antibodies. A real-time RT-PCR assay was performed to detect SARS-CoV-2 RNA. Our findings demonstrated that four captive tigers (6.5%, 4/62) had SARS-CoV-2 neutralizing antibodies against Wuhan Hu-1 and the Delta variant, while no SARS-CoV-2 RNA genome could be detected in all swab samples. Moreover, a low-level titer of neutralizing antibodies against the Omicron BA.2 subvariant could be found in only one seropositive tiger. The source of SARS-CoV-2 infection in these tigers most likely came from close contact with the infected animals’ caretakers who engaged in activities such as tiger petting and feeding. In summary, we described the first case of natural SARS-CoV-2 infection in captive tigers during the COVID-19 outbreak in Thailand and provided seroepidemiological-based evidence of human-to-animal transmission. Our findings highlight the need for continuous surveillance of COVID-19 among the captive tiger population and emphasize the need to adopt a One Health approach for preventing and controlling outbreaks of COVID-19 zoonotic disease.

ABSTRACTAlthoughSalmonella entericacan produce life-threatening colitis in horses, certain serotypes are more commonly associated with clinical disease. Our aim was to evaluate the proportional morbidity attributed to different serotypes, as well as the phenotypic and genotypic antimicrobial resistance (AMR) ofSalmonellaisolates from patients at an equine referral hospital in the southern United States. A total of 255Salmonellaisolates was obtained from clinical samples of patients admitted to the hospital between 2007 and 2015. Phenotypic resistance to 14 antibiotics surveilled by the U.S. National Antimicrobial Resistance Monitoring System was determined using a commercially available panel. Whole-genome sequencing was used to identify serotypes and genotypic AMR. The most common serotypes wereSalmonella entericaserotype Newport (18%),Salmonella entericaserotype Anatum (15.2%), andSalmonella entericaserotype Braenderup (11.8%). Most (n= 219) of the isolates were pansusceptible, while 25 were multidrug resistant (≥3 antimicrobial classes). Genes encoding beta-lactam resistance, such asblaCMY-2,blaSHV-12,blaCTX-M-27, andblaTEM-1B, were detected. TheqnrB2 andaac(6′)-Ib-crgenes were present in isolates with reduced susceptibility to ciprofloxacin. Genes encoding resistance to gentamicin (aph(3′)-Ia,aac(6′)-IIc), streptomycin (strA andstrB), sulfonamides (sul1), trimethoprim (dfrA), phenicols (catA), tetracyclines [tet(A) andtet(E)], and macrolides [ere(A)] were also identified. The main predicted incompatibility plasmid type was I1 (10%). Core genome-based analyses revealed phylogenetic associations between isolates of common serotypes. The presence of AMRSalmonellain equine patients increases the risk of unsuccessful treatment and causes concern for potential zoonotic transmission to attending veterinary personnel, animal caretakers, and horse owners. Understanding the epidemiology ofSalmonellain horses admitted to referral hospitals is important for the prevention, control, and treatment of salmonellosis.IMPORTANCEIn horses, salmonellosis is a leading cause of life-threatening colitis. At veterinary teaching hospitals, nosocomial outbreaks can increase the risk of zoonotic transmission, lead to restrictions on admissions, impact hospital reputation, and interrupt educational activities. The antimicrobials most often used in horses are included in the 5th revision of the World Health Organization's list of critically important antimicrobials for human medicine. Recent studies have demonstrated a trend of increasing bacterial resistance to drugs commonly used to treatSalmonellainfections. In this study, we identify temporal trends in the distribution ofSalmonellaserotypes and their mechanisms of antimicrobial resistance; furthermore, we are able to determine the likely origin of several temporal clusters of infection by using whole-genome sequencing. These data can be used to focus strategies to better contain the dissemination and enhance the mitigation ofSalmonellainfections and to provide evidence-based policies and guidelines to steward antimicrobial use in veterinary medicine.

Our genome is constantly challenged by sources that cause DNA damage. To repair DNA damage and maintain genomic stability eukaryotes have evolved a complex network of pathways termed the DNA damage response (DDR). The DDR consists of signal transduction pathways that sense DNA damage and mediate tightly coordinated reactions to halt the cell cycle and repair DNA with a collection of different enzymes. In this manner, the DDR protects the genome by preventing the accumulation of mutations and DNA aberrations that promote cellular transformation and cancer development. Loss of function mutations in DDR genes and genomic instability occur frequently in many tumor types and underlie numerous cancer-prone hereditary syndromes such as Fanconi Anemia (FA). My thesis research applies candidate-based and unbiased experimental approaches to investigate the role of several tumor suppressor genes (TSGs) in the DDR. My dissertation will first describe a novel function for the breast and ovarian cancer tumor suppressor and FA-associated gene FANCJ in the DDR to ultraviolet (UV) irradiation. In response to UV irradiation FANCJ supports checkpoint induction, the arrest of DNA synthesis, and suppresses UV induced point mutations. Suggesting that FANCJ could suppress UV induced cancers, in sequenced melanomas from multiple databases I found somatic mutations in FANCJ previously associated with breast/ovarian cancer and FA syndrome. The second part of my dissertation will describe an RNA interference screen to identify genes modulating cellular sensitivity to the chemotherapeutic drug cisplatin. The hereditary breast/ovarian cancer tumor suppressor BRCA2 is essential for DNA repair, thus BRCA2 mutant ovarian cancer cells are initially sensitive to cisplatin chemotherapy that induces DNA damage. However, drug resistance develops and remains a major problem in the clinic. My screen identified the chromatin remodeling factor CHD4 as a potent modulator of cisplatin sensitivity and predictor of response to chemotherapy in BRCA2 mutant cancers. Taken together, my investigations highlight the important contribution of the DDR and the role they play in tumorigenesis and predicting therapeutic response.

Our genome is constantly challenged by sources that cause DNA damage. To repair DNA damage and maintain genomic stability eukaryotes have evolved a complex network of pathways termed the DNA damage response (DDR). The DDR consists of signal transduction pathways that sense DNA damage and mediate tightly coordinated reactions to halt the cell cycle and repair DNA with a collection of different enzymes. In this manner, the DDR protects the genome by preventing the accumulation of mutations and DNA aberrations that promote cellular transformation and cancer development. Loss of function mutations in DDR genes and genomic instability occur frequently in many tumor types and underlie numerous cancer-prone hereditary syndromes such as Fanconi Anemia (FA). My thesis research applies candidate-based and unbiased experimental approaches to investigate the role of several tumor suppressor genes (TSGs) in the DDR. My dissertation will first describe a novel function for the breast and ovarian cancer tumor suppressor and FA-associated gene FANCJ in the DDR to ultraviolet (UV) irradiation. In response to UV irradiation FANCJ supports checkpoint induction, the arrest of DNA synthesis, and suppresses UV induced point mutations. Suggesting that FANCJ could suppress UV induced cancers, in sequenced melanomas from multiple databases I found somatic mutations in FANCJ previously associated with breast/ovarian cancer and FA syndrome. The second part of my dissertation will describe an RNA interference screen to identify genes modulating cellular sensitivity to the chemotherapeutic drug cisplatin. The hereditary breast/ovarian cancer tumor suppressor BRCA2 is essential for DNA repair, thus BRCA2 mutant ovarian cancer cells are initially sensitive to cisplatin chemotherapy that induces DNA damage. However, drug resistance develops and remains a major problem in the clinic. My screen identified the chromatin remodeling factor CHD4 as a potent modulator of cisplatin sensitivity and predictor of response to chemotherapy in BRCA2 mutant cancers. Taken together, my investigations highlight the important contribution of the DDR and the role they play in tumorigenesis and predicting therapeutic response.

The focus of my research has been to understand the molecular mechanisms underlying the role of mammalian RAD51 paralogs in genome maintenance and tumor suppression. The investigation carried out during this study has contributed to identify unanticipated functions of these genome caretakers in the cellular response to replication stress and accompanying repair mechanisms. These include maintenance of cellular nucleotide pools through the regulation of ribonucleotide reductase (RNR), modulation of replication fork progression during nucleotide pool alterations and protection of stressed forks to foster genome integrity during perturbed DNA replication. Moreover, my study has uncovered an ATR signaling governed functional interplay between RAD51 paralogs, where distinct paralog complexes collaboratively facilitate genome integrity and cell survival during replication stress

From the simplest single-cellular organism to the most complex multicellular life forms, genetic information in form of DNA represents the universal basis for all biological processes and thus for life itself. Maintaining the structural and functional integrity of the genome is therefore of paramount importance for every single cell. DNA itself, as an active and complex macromolecular structure, is both substrate and product of many of these biochemical processes. A cornerstone of DNA maintenance is thus established by the tight regulation of the multitude of reactions in DNA metabolism, repressing adverse side reactions and ensuring the integrity of DNA in sequence and function. The family of RecQ helicases has emerged as a vital class of enzymes that facilitate genomic integrity by operating in a versatile spectrum of nucleic acid metabolism processes, such as DNA replication, repair, recombination, transcription and telomere stability. RecQ helicases are ubiquitously expressed and conserved in all kingdoms of life. Human cells express five different RecQ enzymes, RecQ1, BLM, WRN, RecQ4 and RecQ5, which all exhibit individual as well as overlapping functions in the maintenance of genomic integrity. Dysfunction of three human RecQ helicases, BLM, WRN and RecQ4, causes different heritable cancer susceptibility syndromes, supporting the theory that genomic instability is a molecular driving force for cancer development. However, based on their inherent DNA protective nature, RecQ helicases represent a double-edged sword in the maintenance of genomic integrity. While their activity in normal cells is essential to prevent cancerogenesis and cellular aging, cancer cells may exploit this DNA protective function by the overexpression of many RecQ helicases, aiding to overcome the disadvantageous results of unchecked DNA replication and simultaneously gaining resistance against chemotherapeutic drugs. Therefore, detailed knowledge how RecQ helicases warrant genomic integrity is required to understand their implication in cancerogenesis and aging, thus setting the stage to develop new strategies towards the treatment of cancer. The current study presents and discusses the first high-resolution X-ray structure of the human RecQ4 helicase. The structure encompasses the conserved RecQ4 helicase core, including a large fraction of its unique C- terminus. Our structural analysis of the RecQ4 model highlights distinctive differences and unexpected similarities to other, structurally conserved, RecQ helicases and permits to draw conclusions about the functional implications of the unique domains within the RecQ4 C-terminus. The biochemical characterization of various RecQ4 variants provides functional insights into the RecQ4 helicase mechanism, suggesting that RecQ4 might utilize an alternative DNA strand separation technique, compared to other human RecQ family members. Finally, the RecQ4 model permits for the first time the analysis of multiple documented RecQ4 patient mutations at the atomic level and thus provides the possibility for an advanced interpretation of particular structure-function relationships in RecQ4 pathogenesis
Vom simpelsten einzelligen Organismus bis hin zu hoch komplexen Lebensformen, genetische Information in Form von DNA repräsentiert die universelle Grundlage aller biologischer Prozesse, und damit die des Lebens selbst. Die Aufrechterhaltung der intakten Struktur und Funktion des Genoms ist daher von höchster Priorität für jede einzelne Zelle. Die DNA selbst, als aktives und komplexes Makromolekül, ist sowohl Substrat als auch Produkt einer Vielzahl dieser biochemischen Prozesse. Ein wesentlicher Aspekt für die Aufrechterhaltung genomischer Integrität besteht daher in der gezielten Regulation aller Prozesse des DNA Metabolismus, um die Konservierung der DNA in Sequenz und Funktion zu gewährleisten und unerwünschte Nebenreaktionen zu verhindern. Die Familie der RecQ Helikasen hat sich als eine essentielle Gruppe von Enzymen etabliert, die diese genomische Integrität gewährleisten, indem sie eine Vielzahl von DNA basierten Prozessen kontrollieren. Dies umfasst die Replikation, Reparatur, Rekombination und Transkription von DNA, sowie Prozesse, die der Stabilisierung der Telomere dienen. RecQ Helikasen werden von allen Zellen exprimiert und können in allen Domänen des Lebens – Bakterien, Archaeen und Eukaryoten nachgewiesen werden. Humane Zellen enthalten fünf verschiedene RecQ Helikasen, RecQ1, BLM, WRN, RecQ4 und RecQ5, welche sowohl individuelle als auch überlappende Funktionen in der Aufrechterhaltung genomischer Integrität innehaben. Eine Beeinträchtigung der Funktion der humanen RecQ Helikasen BLM, WRN und RecQ4 führt zu Krankheiten die durch eine erhöhte Wahrscheinlichkeit für die Entstehung von Krebs gekennzeichnet sind. Dies unterstützt die Theorie, dass die genomische Instabilität eine molekulare Grundlage für die Entstehung von Krebs darstellt. Allerdings repräsentiert die den RecQ Helikasen innewohnende Funktion der Aufrechterhaltung genomischer Integrität ein zweischneidiges Schwert. Während ihre Aktivitäten auf der einen Seite für normale Zellen essentiell sind, um Krankheiten und zelluläre Alterungserscheinungen zu verhindern, wird ihre DNA protektive Funktion von Krebszellen genutzt, indem sie verschiedenste RecQ Helikasen überexprimieren und damit den nachteiligen Effekten der unkontrollierten DNA Replikation entgegenwirken. Zudem erlangen Tumorzellen durch die erhöhte Präsenz der RecQ Helikasen Resistenz gegenüber einer Vielzahl von Chemotherapeutika. Es ist daher von größter Bedeutung zu verstehen, wie genau die einzelnen RecQ Helikasen in der Entstehung von Krebs und dem Alterungsprozess involviert sind, um neue Ansätze in der Krebstherapie zu entwickeln. Die vorliegende Arbeit präsentiert und diskutiert die erste detaillierte Röntgen-Kristallographische Struktur der humanen RecQ4 Helikase. Die vorgestellte Struktur umfasst den konservierten Kern der RecQ4 Helikase, einschließlich eines großen Teils ihres einzigartigen C-terminus. Eine Analyse des RecQ4 Modells weist sowohl eindeutige Unterschiede als auch unerwartete Gemeinsamkeiten im Vergleich mit anderen, untereinander strukturell und funktional ähnlichen, humanen RecQ Helikasen auf und erlaubt zudem Rückschlüsse auf die Funktion der einzigartigen C-terminalen RecQ4 Domäne. Die biochemische Charakterisierung verschiedener RecQ4 Varianten liefert funktionelle Einblicke in den Mechanismus der DNA Doppelstrangtrennung durch RecQ4 und deutet darauf hin, dass sich dieser in weiten Teilen vom Mechanismus der anderen humanen RecQ Helikasen unterscheidet. Letztlich repräsentiert das hier vorgestellte Modell der RecQ4 Helikase die Grundlage für die Analyse verschiedenster dokumentierter RecQ4 Patientenmutationen und erlaubt damit eine erste Abschätzung von Struktur-und-Funktions-Beziehungen bezüglich der bekannten RecQ4- assoziierten Krankheitsbilder