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Frontiers in Cell and Developmental... 2024The accuracy of replication is one of the most important mechanisms ensuring the stability of the genome. The fork protection complex prevents premature replisome...
The accuracy of replication is one of the most important mechanisms ensuring the stability of the genome. The fork protection complex prevents premature replisome stalling and/or premature disassembly upon stress. Here, we characterize the Timeless-Tipin complex, a component of the fork protection complex. We used microscopy approaches, including colocalization analysis and proximity ligation assay, to investigate the spatial localization of the complex during ongoing replication in human cells. Taking advantage of the replication stress induction and the ensuing polymerase-helicase uncoupling, we characterized the Timeless-Tipin localization within the replisome. Replication stress was induced using hydroxyurea (HU) and aphidicolin (APH). While HU depletes the substrate for DNA synthesis, APH binds directly inside the catalytic pocket of DNA polymerase and inhibits its activity. Our data revealed that the Timeless-Tipin complex, independent of the stress, remains bound on chromatin upon stress induction and progresses together with the replicative helicase. This is accompanied by the spatial dissociation of the complex from the blocked replication machinery. Additionally, after stress induction, Timeless interaction with RPA, which continuously accumulates on ssDNA, was increased. Taken together, the Timeless-Tipin complex acts as a universal guardian of the mammalian replisome in an unperturbed S-phase progression as well as during replication stress.
PubMed: 38487270
DOI: 10.3389/fcell.2024.1346534 -
The World Allergy Organization Journal Mar 2024Hereditary angioedema (HAE) is a rare autosomal dominant genetic disease characterised by acute episodes of non-pruritic skin and submucosal swelling caused by increase...
BACKGROUND
Hereditary angioedema (HAE) is a rare autosomal dominant genetic disease characterised by acute episodes of non-pruritic skin and submucosal swelling caused by increase in vascular permeability.
OBJECTIVE
Here we present the first complex analysis of the National HAE Slovakian cohort with the detection of 12 previously un-published genetic variants in gene.
METHODS
In patients diagnosed with hereditary angioedema caused by deficiency or dysfunction of C1 inhibitor (C1-INH-HAE) based on clinical manifestation and complement measurements, gene was tested by DNA sequencing (Sanger sequencing/massive parallel sequencing) and/or multiplex ligation-dependent probe amplification for detection of large rearrangements.
RESULTS
The Slovakian national cohort consisted of 132 living patients with confirmed HAE. We identified 51 index cases (32 families, 19 sporadic patients/112 adults, 20 children). One hundred seventeen patients had HAE caused by deficiency of C1 inhibitor (C1-INH-HAE-1) and 15 patients had HAE caused by dysfunction of C1 inhibitor (C1-INH-HAE-2). The prevalence of HAE in Slovakia has recently been calculated to 1:41 280 which is higher than average calculated prevalence. The estimated incidence was 1:1360 000. Molecular-genetic testing of the gene found 22 unique causal variants in 26 index cases, including 12 previously undescribed and unreported.
CONCLUSION
The first complex report about epidemiology and genetics of the Slovakian national HAE cohort expands the knowledge of the C1-INH-HAE genetics. Twelve novel causal variants were present in the half of the index cases. A higher percentage of inframe variants comparing to other studies was observed. Heterozygous deletion of exon 3 found in a large C1-INH-HAE-1 family probably causes the dysregulation of the splicing isoforms balance and leads to the decrease of full-length C1-INH level.
PubMed: 38486718
DOI: 10.1016/j.waojou.2024.100885 -
STAR Protocols Jun 2024Dinoflagellate genomes often are very large and difficult to assemble, which has until recently precluded their analysis with modern functional genomic tools. Here, we...
Dinoflagellate genomes often are very large and difficult to assemble, which has until recently precluded their analysis with modern functional genomic tools. Here, we present a protocol for mapping three-dimensional (3D) genome organization in dinoflagellates and using it for scaffolding their genome assemblies. We describe steps for crosslinking, nuclear lysis, denaturation, restriction digest, ligation, and DNA shearing and purification. We then detail procedures sequencing library generation and computational analysis, including initial Hi-C read mapping and 3D-DNA scaffolding/assembly correction. For complete details on the use and execution of this protocol, please refer to Marinov et al..
Topics: Dinoflagellida; Genome, Protozoan; Genomics; Chromosome Mapping; Sequence Analysis, DNA
PubMed: 38483898
DOI: 10.1016/j.xpro.2024.102941 -
Frontiers in Microbiology 2024Double-strand breaks (DSBs) are the most dangerous injuries for a genome. When unrepaired, death quickly ensues. In most bacterial systems, DSBs are repaired through...
Double-strand breaks (DSBs) are the most dangerous injuries for a genome. When unrepaired, death quickly ensues. In most bacterial systems, DSBs are repaired through homologous recombination. Nearly one-quarter of bacterial species harbor a second system, allowing direct ligation of broken ends, known as Non-Homologous End Joining (NHEJ). The relative role of both systems in DSBs repair in bacteria has been explored only in a few cases. To evaluate this in the bacterium , we used a modified version of the symbiotic plasmid (264 kb), containing a single copy of the gene. In this plasmid, we inserted an integrative plasmid harboring a modified gene fragment containing an I-SceI site. DSBs were easily inflicted by conjugating a small, replicative plasmid that expresses the I-SceI nuclease into the appropriate strains. Repair of a DSB may be achieved through homologous recombination (either between adjacent or distant repeats) or NHEJ. Characterization of the derivatives that repaired DSB in different configurations, revealed that in most cases (74%), homologous recombination was the prevalent mechanism responsible for repair, with a relatively minor contribution of NHEJ (23%). Inactivation of the I-SceI gene was detected in 3% of the cases. Sequence analysis of repaired derivatives showed the operation of NHEJ. To enhance the number of derivatives repaired through NHEJ, we repeated these experiments in a mutant background. Derivatives showing NHEJ were readily obtained when the DSB occurred on a small, artificial plasmid in a mutant. However, attempts to deliver a DSB on the symbiotic plasmid in a background failed, due to the accumulation of mutations that inactivated the I-SceI gene. This result, coupled with the absence of derivatives that lost the nonessential symbiotic plasmid, may be due to an unusual stability of the symbiotic plasmid, possibly caused by the presence of multiple toxin-antitoxin modules.
PubMed: 38481790
DOI: 10.3389/fmicb.2024.1333194 -
RNA (New York, N.Y.) May 2024N1-methyladenosine (mA) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. mA is generally located in the T loop of cytosolic tRNA...
N1-methyladenosine (mA) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. mA is generally located in the T loop of cytosolic tRNA and between the acceptor and D stems of mitochondrial tRNAs; it is involved in the tertiary interaction that stabilizes tRNA. Human tRNA mA levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure mA, a PCR method to assess mA levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by a qPCR method (TL-qPCR) that measures mA levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template, followed by qPCR using the ligation product as the template. mA interferes with the ligation in specific ways, allowing for the quantitative assessment of mA levels using subnanogram amounts of total RNA. We identify the features of specificity and quantitation for mA-modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.
Topics: RNA, Transfer; Humans; Adenosine; Nucleic Acid Conformation; Real-Time Polymerase Chain Reaction
PubMed: 38471794
DOI: 10.1261/rna.079895.123 -
BMC Neurology Mar 2024Spinal muscular atrophy (SMA) is a rare autosomal recessive hereditary neuromuscular disease caused by survival motor neuron 1 (SMN1) gene deletion or mutation....
Optimized MLPA workflow for spinal muscular atrophy diagnosis: identification of a novel variant, NC_000005.10:g.(70919941_70927324)del in isolated exon 1 of SMN1 gene through long-range PCR.
BACKGROUND
Spinal muscular atrophy (SMA) is a rare autosomal recessive hereditary neuromuscular disease caused by survival motor neuron 1 (SMN1) gene deletion or mutation. Homozygous deletions of exon 7 in SMN1 result in 95% of SMA cases, while the remaining 5% are caused by other pathogenic variants of SMN1.
METHODS
We analyzed two SMA-suspected cases that were collected, with no SMN1 gene deletion and point mutation in whole-exome sequencing. Exon 1 deletion of the SMN gene was detected using Multiplex ligation-dependent probe amplification (MLPA) P021. We used long-range polymerase chain reaction (PCR) to isolate the SMN1 template, optimized-MLPA P021 for copy number variation (CNV) analysis within SMN1 only, and validated the findings via third-generation sequencing.
RESULTS
Two unrelated families shared a genotype with one copy of exon 7 and a novel variant, g.70919941_70927324del, in isolated exon 1 of the SMN1 gene. Case F1-II.1 demonstrated no exon 1 but retained other exons, whereas F2-II.1 had an exon 1 deletion in a single SMN1 gene. The read coverage in the third-generation sequencing results of both F1-II.1 and F2-II.1 revealed a deletion of approximately 7.3 kb in the 5' region of SMN1. The first nucleotide in the sequence data aligned to the 7385 bp of NG_008691.1.
CONCLUSION
Remarkably, two proband families demonstrated identical SMN1 exon 1 breakpoint sites, hinting at a potential novel mutation hotspot in Chinese SMA, expanding the variation spectrum of the SMN1 gene and corroborating the specificity of isolated exon 1 deletion in SMA pathogenesis. The optimized-MLPA P021 determined a novel variant (g.70919941_70927324del) in isolated exon 1 of the SMN1 gene based on long-range PCR, enabling efficient and affordable detection of SMN gene variations in patients with SMA, providing new insight into SMA diagnosis to SMN1 deficiency and an optimized workflow for single exon CNV testing of the SMN gene.
Topics: Humans; Multiplex Polymerase Chain Reaction; DNA Copy Number Variations; Workflow; Muscular Atrophy, Spinal; Motor Neurons; Exons; Survival of Motor Neuron 1 Protein
PubMed: 38468256
DOI: 10.1186/s12883-024-03592-5 -
Annals of Pediatric Endocrinology &... Feb 2024Deficiency of 21-hydroxylase (21-OHD) is an autosomal recessively inherited disorder that is characterized by adrenal insufficiency and androgen excess. This study was...
PURPOSE
Deficiency of 21-hydroxylase (21-OHD) is an autosomal recessively inherited disorder that is characterized by adrenal insufficiency and androgen excess. This study was performed to investigate the clinical utility of prenatal diagnosis of 21-OHD using molecular genetic testing in families at risk.
METHODS
This study included 27 pregnant women who had previously borne a child with 21-OHD. Fetal tissues were obtained using chorionic villus sampling (CVS) or amniocentesis. After the genomic DNA was isolated, Sanger sequencing of CYP21A2 and multiplex ligation-dependent probe amplification were performed. The clinical and endocrinological findings were reviewed retrospectively.
RESULTS
A total of 39 prenatal genetic tests was performed on 27 pregnant women and their fetal tissues. The mean gestational age at the time of testing was 11.7 weeks for CVS and 17.5 weeks for amniocentesis. Eleven fetuses (28.2%) were diagnosed with 21-OHD. Among them, 10 fetuses (90.9%) harbored the same mutation as siblings who were previously diagnosed with 21-OHD. Among these, 4 fetuses (3 males and 1 female) identified as affected were born alive. All 4 patients have been treated with hydrocortisone, 9α-fludrocortisone, and sodium chloride since a mean of 3.7 days of life. The male patients did not show hyponatremia and dehydration, although they harbored pathogenic variants associated with the salt-wasting type of 21-OHD.
CONCLUSION
This study demonstrated the diagnostic efficacy and clinical consequences of diagnosis by prenatal genetic testing in families at risk for 21-OHD. All patients identified as affected were treated with hydrocortisone and 9α-fludrocortisone early after birth, which can prevent a life-threatening adrenal crisis.
PubMed: 38461806
DOI: 10.6065/apem.2346014.007 -
Journal of Radiation Research May 2024Ionizing radiation (IR)-induced double-strand breaks (DSBs) are primarily repaired by non-homologous end joining or homologous recombination (HR) in human cells. DSB...
Ionizing radiation (IR)-induced double-strand breaks (DSBs) are primarily repaired by non-homologous end joining or homologous recombination (HR) in human cells. DSB repair requires adenosine-5'-triphosphate (ATP) for protein kinase activities in the multiple steps of DSB repair, such as DNA ligation, chromatin remodeling, and DNA damage signaling via protein kinase and ATPase activities. To investigate whether low ATP culture conditions affect the recruitment of repair proteins at DSB sites, IR-induced foci were examined in the presence of ATP synthesis inhibitors. We found that p53 binding protein 1 foci formation was modestly reduced under low ATP conditions after IR, although phosphorylated histone H2AX and mediator of DNA damage checkpoint 1 foci formation were not impaired. Next, we examined the foci formation of breast cancer susceptibility gene I (BRCA1), replication protein A (RPA) and radiation 51 (RAD51), which are HR factors, in G2 phase cells following IR. Interestingly, BRCA1 and RPA foci in the G2 phase were significantly reduced under low ATP conditions compared to that under normal culture conditions. Notably, RAD51 foci were drastically impaired under low ATP conditions. These results suggest that HR does not effectively progress under low ATP conditions; in particular, ATP shortages impair downstream steps in HR, such as RAD51 loading. Taken together, these results suggest that the maintenance of cellular ATP levels is critical for DNA damage response and HR progression after IR.
Topics: Humans; Adenosine Triphosphate; Homologous Recombination; Radiation, Ionizing; Rad51 Recombinase; BRCA1 Protein; DNA Breaks, Double-Stranded; Replication Protein A; Cell Line, Tumor; Intracellular Space; DNA Repair; Histones
PubMed: 38461549
DOI: 10.1093/jrr/rrae005 -
Nature Communications Mar 2024Fine-mapping and functional studies implicate rs117701653, a non-coding single nucleotide polymorphism in the CD28/CTLA4/ICOS locus, as a risk variant for rheumatoid...
Fine-mapping and functional studies implicate rs117701653, a non-coding single nucleotide polymorphism in the CD28/CTLA4/ICOS locus, as a risk variant for rheumatoid arthritis and type 1 diabetes. Here, using DNA pulldown, mass spectrometry, genome editing and eQTL analysis, we establish that the disease-associated risk allele is functional, reducing affinity for the inhibitory chromosomal regulator SMCHD1 to enhance expression of inducible T-cell costimulator (ICOS) in memory CD4 T cells from healthy donors. Higher ICOS expression is paralleled by an increase in circulating T peripheral helper (Tph) cells and, in rheumatoid arthritis patients, of blood and joint fluid Tph cells as well as circulating plasmablasts. Correspondingly, ICOS ligation and carriage of the rs117701653 risk allele accelerate T cell differentiation into CXCR5PD-1 Tph cells producing IL-21 and CXCL13. Thus, mechanistic dissection of a functional non-coding variant in human autoimmunity discloses a previously undefined pathway through which ICOS regulates Tph development and abundance.
Topics: Humans; T-Lymphocytes; Arthritis, Rheumatoid; Inducible T-Cell Co-Stimulator Protein; CD28 Antigens; Alleles; T-Lymphocytes, Helper-Inducer; Chromosomal Proteins, Non-Histone
PubMed: 38459032
DOI: 10.1038/s41467-024-46457-8 -
Scientific Reports Mar 2024In this report, we present OLAF-Seq, a novel strategy to construct a long-read sequencing library such that adjacent fragments are linked with end-terminal duplications....
In this report, we present OLAF-Seq, a novel strategy to construct a long-read sequencing library such that adjacent fragments are linked with end-terminal duplications. We use the CRISPR-Cas9 nickase enzyme and a pool of multiple sgRNAs to perform non-random fragmentation of targeted long DNA molecules (> 300kb) into smaller library-sized fragments (about 20 kbp) in a manner so as to retain physical linkage information (up to 1000 bp) between adjacent fragments. DNA molecules targeted for fragmentation are preferentially ligated with adaptors for sequencing, so this method can enrich targeted regions while taking advantage of the long-read sequencing platforms. This enables the sequencing of target regions with significantly lower total coverage, and the genome sequence within linker regions provides information for assembly and phasing. We demonstrated the validity and efficacy of the method first using phage and then by sequencing a panel of 100 full-length cancer-related genes (including both exons and introns) in the human genome. When the designed linkers contained heterozygous genetic variants, long haplotypes could be established. This sequencing strategy can be readily applied in both PacBio and Oxford Nanopore platforms for both long and short genes with an easy protocol. This economically viable approach is useful for targeted enrichment of hundreds of target genomic regions and where long no-gap contigs need deep sequencing.
Topics: Humans; RNA, Guide, CRISPR-Cas Systems; Sequence Analysis, DNA; Genomics; Bacteriophages; CRISPR-Associated Protein 9; DNA
PubMed: 38448490
DOI: 10.1038/s41598-024-56402-w