Animals ; CRISPR-Cas Systems ; Endodeoxyribonucleases ; genetics ; metabolism ; Gene Editing ; methods ; Mice ; Transcription Activator-Like Effector Nucleases ; genetics ; metabolism

Trinucleotide repeat (TNR) instability can cause a variety of human genetic diseases including myotonic dystrophy and Huntington's disease. Recent genetic data show that instability of the CAG/CTG repeat DNA is dependent on its length and replication origin. In yeast, the RAD27 (human FEN-1 homologue) null mutant has a high expansion frequency at the TNR loci. We demonstrate here that FEN-1 processes the 5'-flap DNA of CTG/CAG repeats, which is dependent on the length in vitro. FEN-1 protein can cleave the 5'-flap DNA containing triplet repeating sequence up to 21 repeats, but the activity decreases with increasing size of flap above 11 repeats. In addition, FEN-1 processing of 5'-flap DNA depends on sequence, which play a role in the replication origin-dependent TNR instability. Interestingly, FEN-1 can cleave the 5'-flap DNA of CTG repeats better than CAG repeats possibly through the flap-structure. Our biochemical data of FEN-1's activity with triplet repeat DNA clearly shows length dependence, and aids our understanding on the mechanism of TNR instability.
Base Sequence ; DNA, Single-Stranded/*metabolism ; Endodeoxyribonucleases/genetics/*metabolism ; Flap Endonucleases ; Gene Expression Regulation ; Genetic Diseases, Inborn/*genetics ; Human ; Nucleic Acid Conformation ; Trinucleotide Repeat Expansion ; *Trinucleotide Repeats

Trinucleotide repeat (TNR) instability can cause a variety of human genetic diseases including myotonic dystrophy and Huntington's disease. Recent genetic data show that instability of the CAG/CTG repeat DNA is dependent on its length and replication origin. In yeast, the RAD27 (human FEN-1 homologue) null mutant has a high expansion frequency at the TNR loci. We demonstrate here that FEN-1 processes the 5'-flap DNA of CTG/CAG repeats, which is dependent on the length in vitro. FEN-1 protein can cleave the 5'-flap DNA containing triplet repeating sequence up to 21 repeats, but the activity decreases with increasing size of flap above 11 repeats. In addition, FEN-1 processing of 5'-flap DNA depends on sequence, which play a role in the replication origin-dependent TNR instability. Interestingly, FEN-1 can cleave the 5'-flap DNA of CTG repeats better than CAG repeats possibly through the flap-structure. Our biochemical data of FEN-1's activity with triplet repeat DNA clearly shows length dependence, and aids our understanding on the mechanism of TNR instability.
Base Sequence ; DNA, Single-Stranded/*metabolism ; Endodeoxyribonucleases/genetics/*metabolism ; Flap Endonucleases ; Gene Expression Regulation ; Genetic Diseases, Inborn/*genetics ; Human ; Nucleic Acid Conformation ; Trinucleotide Repeat Expansion ; *Trinucleotide Repeats

Objective: To report the clinical features and genetic variations of monogenic lupus caused by DNASE1L3 deficiency and to introduce preliminary experience on diagnosis and treatment for this disease. Methods: Clinical data of 3 children from the same pedigree were collected who were diagnosed with DNASE1L3 defect-associated monogenic lupus in August 2020 by Department of Pediatrics, Peking Union Medical College Hospital referred from Department of Pediatrics, Boai Hospital of Zhongshan. DNA was extracted from the peripheral blood of the patients and their parients to perform genetic analysis and confirmation. Six interferon-stimulated genes were relatively quantified to examine the activation of the type I interferon signaling. "DNASE1L3" "systemic lupus erythematosus" and "SLE" were searched in PubMed, Wangfang Data, CNKI databases for related reports from database established date to June 2022. Spectrum of genetic variations and clinical phenotypes were analyzed in combination with this pedigree. Results: Case 1, a 14-year-old girl with edema, hematuria, and heavy proteinuria, presented with membranous nephropathy. Case 2, the 12-year-old younger brother of case 1 with hematologic, cardiac, pulmonary, renal involvement, positive antinuclear antibody, positive anti-double-stranded DNA antibody and low complement C3, manifested with systemic lupus erythematosus. Case 3, the 8-year-old younger sister of case 1 with hematologic, cardiac, pulmonary and renal involvement, positive antinuclear antibody, positive anti-double-stranded DNA antibody, and low complement C3 and C4, manifested with systemic lupus erythematosus. Genetic testing revealed that all 3 patients carried homozygous deletions in exons 3 and 4 on DNASE1L3 gene. Interferon scores were elevated in case 1, 2 and their parents but normal in case 3. All 3 patients were diagnosed with monogenic lupus caused by DNASE1L3 defects. Literature searching identified 10 relevant publications in English and 0 publication in Chinese, involving 42 patients from 18 pedigrees (including the 3 cases from this pedigree). Nine variants were found: c.289_290delAC (p.T97Ifs*2), c.643delT (p.W215Gfs*2), c.320+4delAGTA, c.321-1G>A, Ex5 del, c.433G>A, c.581G>A (p.C194Y), c.537G>A (p.W179X), and Ex3-4 del. The hotspot variants were c.643delT (43% (36/84)) and c.289_290delAC (36% (30/84)). Kidney was affected in 31 cases (74%) of the 42 cases. Among the 25 patients, joints were affected in 16 cases (64%), fever were reported in 13 cases (52%) hematologic system was involved 13 cases (52%), rash was present in 10 cases (40%), intestinal tract was involved in 8 cases (32%), lungs were involved in 6 cases (24%), eyes were involved in 4 cases (16%), and the heart was involved in 4 cases (16%). The 2 cardiopulmonary affected patients from literature showed poor prognosis, with 1 died, and 1 right heart failure. Conclusions: The clinical manifestations of monogenic lupus caused by DNASE1L3 defect are highly heterogenous, primarily with renal, blood, joint, intestinal, and cardiopulmonary involvement. There is no correlation between the genotype and the phenotype. DNASE1L3 defects were predominantly mediated by null varations including nonsense, splicing, frameshift and exon deletions. The hotspot variants are c.643delT and c.289_290delAC. DNASE1L3 defects should be cautioned in early-onset lupus-like patients with renal, joint and hematologic involvement. Cardiopulmonary involved patients require close monitoring for poor prognosis. Copy number variations should be carefully analyzed after negative whole exome sequencing.
Male ; Child ; Humans ; Homozygote ; Complement C3 ; Antibodies, Antinuclear ; DNA Copy Number Variations ; Sequence Deletion ; Interferons ; Lupus Erythematosus, Systemic/genetics* ; Antiviral Agents ; Endodeoxyribonucleases

To efficiently produce non-specific nuclease (NU) of Serratia marcescens through recombinant overexpression approach and to characterize the purified NU. The nuclease gene was amplified from the genomic DNA of Serratia marcescens by PCR and fused into vector pMAL-c4X with maltose binding protein (MBP) tag. The recombinant vector verified by DNA sequencing was transformed into Escherichia coli BL21. The expressed MBP-NU was purified through the amylose resin and its catalytic characters were analyzed. The results showed the NU gene had 97% identities with the reported S. marcescens nuclease gene and intracellularly expressed in E. coli BL21. The optimal expression conditions were 37 degrees C, 0.75 mmol/L IPTG with 1.5 h induction. The purified MBP-NU exhibited non-specific nuclease activity, able to degrade various nucleic acids, including RNA, single-stranded DNA and double-stranded DNA that was circular or linear. Its optimal temperature was 37 degrees C and optimal pH 8.0. From 1 L culture broth 10.8 mg NU could be purified with a specific activity of 1.11x10(6) U/mg. The catalytic activity of NU was not inhibited by reagents such as EDTA (0.5 mmol/L), PMSF (1 mmol/L) and KCl (150 mmol/L) commonly used in protein purification.
Base Sequence ; Endodeoxyribonucleases ; biosynthesis ; genetics ; Endoribonucleases ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Maltose-Binding Proteins ; genetics ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Serratia marcescens ; enzymology

Flap endo/exonuclease-1 (FEN-1) recognizes 5'-flap DNA structures that have been proposed to be important intermediates in DNA replication, repair and recombination, and cleaves the double strand-single strand junction of flap substrates. Using an in vitro model system, recent studies have shown that FEN-1 is a necessary enzyme for the removal of RNA primers in Okazaki fragment maturation during lagging strand DNA synthesis. In this report, the FEN-1 gene expression was examined during cell cycle and differentiation. Although FEN-1 mRNA and protein could be detected at all stages of the cell cycle, their levels were more elevated in exponentially proliferating cells than in G1 or G2/M-synchronized cells. Moreover, a significant increase of FEN-1 protein was observed when temporarily quiescent fibroblasts were induced to proliferate by serum stimulation. In contrast, the FEN-1 mRNA level showed a sharp decrease in HL-60 cells differentiated by dimethyl-sulfoxide, all-trans retinoic acid or 12-O-tetradecanoylphorbol-13-acetate. These results demonstrate that the FEN-1 gene expression is up-regulated during entrance into the mitotic cell cycle and down-regulated in nongrowing cells, as in the case of differentiated promyelocytic leukemia cells.
3T3 Cells ; Animal ; Blotting, Western ; Cell Cycle/genetics ; Cell Differentiation ; Cell Division/genetics* ; Dimethyl Sulfoxide/pharmacology ; Down-Regulation (Physiology) ; Endodeoxyribonucleases/genetics* ; Flow Cytometry ; Gene Expression Regulation, Neoplastic* ; HL-60 Cells ; Human ; Leukemia, Promyelocytic, Acute/genetics* ; Mice

OBJECTIVETo explore the possible roles of polymorphisms of SPO11 and glutathionine S-transferase (GST) genes in idiopathic male infertility in a ethnic Han Chinese population from Henan.

METHODSMultiplex PCR and DNA sequencing were performed to determine the SPO11 c.517C>T(rs28368082) and GST genes (GSTM1, GSTT1, GSTP1) polymorphisms in 216 idiopathic male infertility cases and 198 normal samples.

RESULTSThe frequencies of the SPO11 CC and CT genotypes were 87.5% (189/216) and 12.5% (27/216) in the patients, and 97.5% (193/198) and 2.5% (5/198) in the controls, respectively. The frequencies of SPO11 CC and CT genotypes, the A>G transition at nucleotide 313 in the exon 5 of the GSTP1 gene, and the frequencies of combined genotypes GSTM1 (-/-), GSTT1 (+/+), GSTP1 (AA) and SPO11 (CT) were significantly different between the two groups (P<0.05).

CONCLUSIONThe rs28368082 polymorphism of the SPO11 gene, the A>G transition at nucleotide 313 in the exon 5 of the GSTP1 gene, and the combined genotypes of GSTM1 (-/-), GSTT1 (+/+), GSTP1 (AA) and SPO11 (CT) may be associated with idiopathic male infertility in ethnic Han Chinese.

Adult ; Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Endodeoxyribonucleases ; genetics ; Gene Frequency ; Genetic Predisposition to Disease ; ethnology ; genetics ; Genotype ; Glutathione S-Transferase pi ; genetics ; Glutathione Transferase ; genetics ; Humans ; Infertility, Male ; enzymology ; ethnology ; genetics ; Linkage Disequilibrium ; Male ; Mutation ; Odds Ratio ; Polymorphism, Genetic ; Sequence Analysis, DNA

Meiotic recombination is carried out through a specialized pathway for the formation and repair of DNA double-strand breaks (DSBs) made by the Spo11 protein. The present study shed light on the functional role of cyclin, CYC2, in Tetrahymena thermophila which has transcriptionally high expression level during meiosis process. Knocking out the CYC2 gene results in arrest of meiotic conjugation process at 2.5-3.5 h after conjugation initiation, before the meiosis division starts, and in company with the absence of DSBs. To investigate the underlying mechanism of this phenomenon, a complete transcriptome profile was performed between wild-type strain and CYC2 knock-out strain. Functional analysis of RNA-Seq results identifies related differentially expressed genes (DEGs) including SPO11 and these DEGs are enriched in DNA repair/mismatch repair (MMR) terms in homologous recombination (HR), which indicates that CYC2 could play a crucial role in meiosis by regulating SPO11 and participating in HR.
Cell Cycle Checkpoints ; Cyclins ; genetics ; metabolism ; DNA Breaks, Double-Stranded ; DNA Mismatch Repair ; DNA Repair ; Endodeoxyribonucleases ; genetics ; metabolism ; Homologous Recombination ; Meiosis ; Microscopy, Fluorescence ; Phenotype ; Protozoan Proteins ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, RNA ; Tetrahymena thermophila ; genetics ; metabolism ; Transcriptome