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Antibiotics (Basel, Switzerland) Jun 2024The emergence of carbapenem-resistant Gram-negative pathogens presents a clinical challenge in infection treatment, prompting the repurposing of existing drugs as an...
The emergence of carbapenem-resistant Gram-negative pathogens presents a clinical challenge in infection treatment, prompting the repurposing of existing drugs as an essential strategy to address this crisis. Although the anticancer drug 5-fluorouracil (5-FU) has been recognized for its antibacterial properties, its mechanisms are not fully understood. Here, we found that the minimal inhibitory concentration (MIC) of 5-FU against was 32-64 µg/mL, including strains carrying , which confers resistance to carbapenems. We further elucidated the antibacterial mechanism of 5-FU against by using genetic and biochemical analyses. We revealed that the mutation of uracil phosphoribosyltransferase-encoding gene increased the MIC of 5-FU against by 32-fold, indicating the role of the gene in 5-FU resistance. Additionally, transcriptomic analysis of treated with 5-FU at 8 µg/mL and 32 µg/mL identified 602 and 1082 differentially expressed genes involved in carbon and nucleic acid metabolism, DNA replication, and repair pathways. The biochemical assays showed that 5-FU induced bacterial DNA damage, significantly increased intracellular ATP levels and the NAD/NADH ratio, and promoted reactive oxygen species (ROS) production. These findings suggested that 5-FU may exert antibacterial effects on through multiple pathways, laying the groundwork for its further development as a therapeutic candidate against carbapenem-resistant bacterial infections.
PubMed: 38927194
DOI: 10.3390/antibiotics13060528 -
Biomolecules Jun 2024Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for...
Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for bionanotechology applications. Although clickable dNTPs are easily incorporated by DNA polymerases into the growing chain, afterwards they might become targets for DNA repair systems or interfere with faithful nucleotide insertion. Little is known about the possibility and mechanisms of these post-synthetic events. Here, we investigated the repair and (mis)coding properties of EtU and two bulkier clickable pyrimidine nucleosides, 5-(octa-1,7-diyn-1-yl)-U (C8-AlkU) and 5-(octa-1,7-diyn-1-yl)-C (C8-AlkC). In vitro, EtU and C8-AlkU, but not C8-AlkC, were excised by SMUG1 and MBD4, two DNA glycosylases from the base excision repair pathway. However, when placed into a plasmid encoding a fluorescent reporter inactivated by repair in human cells, EtU and C8-AlkU persisted for much longer than uracil or its poorly repairable phosphorothioate-flanked derivative. DNA polymerases from four different structural families preferentially bypassed EtU, C8-AlkU and C8-AlkC in an error-free manner, but a certain degree of misincorporation was also observed, especially evident for DNA polymerase β. Overall, clickable pyrimidine nucleotides could undergo repair and be a source of mutations, but the frequency of such events in the cell is unlikely to be considerable.
Topics: DNA Repair; Humans; Pyrimidine Nucleotides; Click Chemistry; DNA-Directed DNA Polymerase; Deoxyuridine; DNA; DNA Replication; Uracil-DNA Glycosidase
PubMed: 38927084
DOI: 10.3390/biom14060681 -
Zhongguo Shi Yan Xue Ye Xue Za Zhi Jun 2024Studies have found that 1/3 patients with acquired aplastic anemia have shortened telomere length, and the shorter the telomere, the longer the disease course, the more... (Review)
Review
Studies have found that 1/3 patients with acquired aplastic anemia have shortened telomere length, and the shorter the telomere, the longer the disease course, the more prone to relapse, the lower the overall survival rate, and the higher the probability of clonal evolution. The regulation of telomere length is affected by many factors, including telomerase activity, telomerase-related genes, telomere regulatory proteins and other related factors. Telomere shortening can lead to genetic instability and increases the probability of clonal evolution in patients with acquired aplastic anemia. This article reviews the role of telomere in the clonal evolution of acquired aplastic anemia and factors affecting telomere length.
Topics: Anemia, Aplastic; Telomere Shortening; Clonal Evolution; Survival Rate; Recurrence; Telomere Homeostasis; Telomerase; Genomic Instability; Humans
PubMed: 38926996
DOI: 10.19746/j.cnki.issn.1009-2137.2024.03.048 -
Nature Communications Jun 2024Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR),...
Oncogene-induced senescence (OIS) arrests cell proliferation in response to replication stress (RS) induced by oncogenes. OIS depends on the DNA damage response (DDR), but also on the cGAS-STING pathway, which detects cytosolic DNA and induces type I interferons (IFNs). Whether and how RS and IFN responses cooperate to promote OIS remains unknown. Here, we show that the induction of OIS by the H-RAS oncogene in immortalized human fibroblasts depends on the MRE11 nuclease. Indeed, treatment with the MRE11 inhibitor Mirin prevented RS, micronuclei formation and IFN response induced by RAS. Overexpression of the cytosolic nuclease TREX1 also prevented OIS. Conversely, overexpression of a dominant negative mutant of TREX1 or treatment with IFN-β was sufficient to induce RS and DNA damage, independent of RAS induction. These data suggest that the IFN response acts as a positive feedback loop to amplify DDR in OIS through a process regulated by MRE11 and TREX1.
Topics: Humans; Exodeoxyribonucleases; Phosphoproteins; MRE11 Homologue Protein; Signal Transduction; Cellular Senescence; DNA Replication; DNA Damage; Fibroblasts; Interferon-beta
PubMed: 38926338
DOI: 10.1038/s41467-024-49740-w -
Discovery Medicine Jun 2024In recent years, a gene-editing technology known as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been developed and is progressively...
BACKGROUND
In recent years, a gene-editing technology known as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been developed and is progressively advancing into clinical trials. While current antiviral therapies are unable to eliminate the Hepatitis B virus (HBV), it stands as a prime target for the CRISPR/Cas9 technology. The objective of this study was to enhance the efficacy of CRISPR/Cas9 in suppressing HBV replication, lowering HBsAg and HBeAg levels, and eliminating covalently closed circular DNA (cccDNA).
METHODS
To enhance the anti-HBV effectiveness of CRISPR/Cas9, our study delved into a dual-guide RNA (gRNA) strategy. After evaluating the antiviral activities of multiple gRNAs that effectively impeded HBV replication, we identified three specific gRNAs-namely 10, 4, and 21. These gRNAs were selected for their targeting of distinct yet conserved regions within the HBV genome.
RESULTS
In HBV-stable cell lines, namely HepAD38, and HBV infection models of HepG2-NTCP cells, our investigation revealed that the co-application of gRNA-10 with either gRNA-4 or gRNA-21 within the CRISPR/Cas9 system demonstrated heightened efficacy in impeding HBV replication, reducing the levels of HBsAg, HBeAg, and cccDNA levels, along with a more pronounced promotion of HBsAg clearance when compared to the use of a single gRNA.
CONCLUSIONS
The CRISPR/Cas9 system employing dual gRNAs has proven highly effective in both suppressing HBV replication and facilitating HBsAg clearance. This promising outcome suggests that it holds potential to emerge as a novel approach for achieving the functional cure of patients with HBV infection.
Topics: Hepatitis B virus; Humans; Virus Replication; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Hep G2 Cells; Gene Editing; DNA, Circular; DNA, Viral; Hepatitis B Surface Antigens; Hepatitis B e Antigens; Antiviral Agents; Hepatitis B
PubMed: 38926103
DOI: 10.24976/Discov.Med.202436185.107 -
The Journal of Molecular Diagnostics :... Jun 2024Replication-coupled gene editing using locked-nucleic-acid-modified single-stranded oligodeoxyribonucleotides (LMOs) can genetically engineer mammalian cells with high...
Replication-coupled gene editing using locked-nucleic-acid-modified single-stranded oligodeoxyribonucleotides (LMOs) can genetically engineer mammalian cells with high precision at single nucleotide resolution. Based on this method, we developed oligonucleotide-directed mutation screening (ODMS) to determine whether variants of uncertain clinical significance of DNA mismatch-repair (MMR) genes can cause Lynch syndrome. In ODMS, the appearance of 6-thioguanine (6TG)-resistant colonies upon introduction of the variant is indicative for defective MMR and hence pathogenicity. Whereas previously mouse embryonic stem cells (mESCs) hemizygous for DNA mismatch-repair (MMR) genes were used, we now show that ODMS can also be applied in wild-type mESCs carrying two functional alleles of each MMR gene. 6TG resistance can result from two possible events: first, the mutation is present in only one allele, which is indicative for dominant-negative activity of the variant; second, both alleles contain the planned modification, which is indicative for a regular loss-of-function variant. Thus, ODMS in wild-type mESCs can discriminate fully disruptive and dominant-negative MMR variants. The feasibility of biallelic targeting suggested that the efficiency of LMO-mediated gene targeting at a non-selectable locus may be enriched in cells that had undergone a simultaneous selectable LMO targeting event. This turned out to be the case and provided a protocol to improve recovery of LMO-mediated gene modification events.
PubMed: 38925454
DOI: 10.1016/j.jmoldx.2024.05.011 -
Molecular Cell Jun 2024The evolutionarily conserved HIRA/Hir histone chaperone complex and ASF1a/Asf1 co-chaperone cooperate to deposit histone (H3/H4) tetramers on DNA for...
The evolutionarily conserved HIRA/Hir histone chaperone complex and ASF1a/Asf1 co-chaperone cooperate to deposit histone (H3/H4) tetramers on DNA for replication-independent chromatin assembly. The molecular architecture of the HIRA/Hir complex and its mode of histone deposition have remained unknown. Here, we report the cryo-EM structure of the S. cerevisiae Hir complex with Asf1/H3/H4 at 2.9-6.8 Å resolution. We find that the Hir complex forms an arc-shaped dimer with a Hir1/Hir2/Hir3/Hpc2 stoichiometry of 2/4/2/4. The core of the complex containing two Hir1/Hir2/Hir2 trimers and N-terminal segments of Hir3 forms a central cavity containing two copies of Hpc2, with one engaged by Asf1/H3/H4, in a suitable position to accommodate a histone (H3/H4) tetramer, while the C-terminal segments of Hir3 harbor nucleic acid binding activity to wrap DNA around the Hpc2-assisted histone tetramer. The structure suggests a model for how the Hir/Asf1 complex promotes the formation of histone tetramers for their subsequent deposition onto DNA.
PubMed: 38925115
DOI: 10.1016/j.molcel.2024.05.031 -
Proceedings of the National Academy of... Jul 2024Maintenance of DNA integrity is essential to all forms of life. DNA damage generated by reaction with genotoxic chemicals results in deleterious mutations, genome...
Maintenance of DNA integrity is essential to all forms of life. DNA damage generated by reaction with genotoxic chemicals results in deleterious mutations, genome instability, and cell death. Pathogenic bacteria encounter several genotoxic agents during infection. In keeping with this, the loss of DNA repair networks results in virulence attenuation in several bacterial species. Interstrand DNA crosslinks (ICLs) are a type of DNA lesion formed by covalent linkage of opposing DNA strands and are particularly toxic as they interfere with replication and transcription. Bacteria have evolved specialized DNA glycosylases that unhook ICLs, thereby initiating their repair. In this study, we describe AlkX, a DNA glycosylase encoded by the multidrug resistant pathogen . AlkX exhibits ICL unhooking activity similar to that of its homolog YcaQ. Interrogation of the in vivo role of AlkX revealed that its loss sensitizes cells to DNA crosslinking and impairs colonization of the lungs and dissemination to distal tissues during pneumonia. These results suggest that AlkX participates in pathogenesis and protects the bacterium from stress conditions encountered in vivo. Consistent with this, we found that acidic pH, an environment encountered during host colonization, results in DNA damage and that a is induced by, and contributes to, defense against acidic conditions. Collectively, these studies reveal functions for a recently described class of proteins encoded in a broad range of pathogenic bacterial species.
Topics: Acinetobacter baumannii; DNA Glycosylases; DNA Damage; DNA Repair; Acinetobacter Infections; Bacterial Proteins; Animals; Mice; DNA, Bacterial; Virulence; Escherichia coli
PubMed: 38923984
DOI: 10.1073/pnas.2402422121 -
Journal of Medical Virology Jun 2024Functional cure of hepatitis B virus (HBV) is an optimal treatment goal for chronic hepatitis B, with the loss of hepatitis B surface antigen (HBsAg) being a crucial...
Functional cure of hepatitis B virus (HBV) is an optimal treatment goal for chronic hepatitis B, with the loss of hepatitis B surface antigen (HBsAg) being a crucial indicator. However, the adequacy of HBsAg loss for evaluating functional cure of HBV in patients co-infected with HBV/human immunodeficiency virus (HIV) remains controversial. In this study, we measured HBV pregenomic RNA (pgRNA), a potential biomarker that correlates with covalently closed circular DNA, in the frozen plasma of 98 patients with HBsAg loss from a large HIV/HBV co-infection cohort in Guangzhou, China. HBV pgRNA was still detected in 43.9% (44/98) of the patients, suggesting active HBV replication in individuals with HBsAg loss. Our observations imply that HBsAg loss may not be a reliable predictor of HBV functional cure in cases of HIV/HBV co-infection.
Topics: Humans; HIV Infections; Hepatitis B Surface Antigens; Coinfection; Male; Hepatitis B virus; Female; Adult; RNA, Viral; Biomarkers; Middle Aged; Hepatitis B, Chronic; China; DNA, Viral; Virus Replication; Cohort Studies; RNA
PubMed: 38923563
DOI: 10.1002/jmv.29762 -
Molecular Microbiology Jun 2024In every bacterium, nucleoid-associated proteins (NAPs) play crucial roles in chromosome organization, replication, repair, gene expression, and other DNA transactions.... (Review)
Review
In every bacterium, nucleoid-associated proteins (NAPs) play crucial roles in chromosome organization, replication, repair, gene expression, and other DNA transactions. Their central role in controlling the chromatin dynamics and transcription has been well-appreciated in several well-studied organisms. Here, we review the diversity, distribution, structure, and function of NAPs from the genus Mycobacterium. We highlight the progress made in our understanding of the effects of these proteins on various processes and in responding to environmental stimuli and stress of mycobacteria in their free-living as well as during distinctive intracellular lifestyles. We project them as potential drug targets and discuss future studies to bridge the information gap with NAPs from well-studied systems.
PubMed: 38922783
DOI: 10.1111/mmi.15287