Pif1 subfamily helicase is conserved from yeast to humans with a lot of cellular functions. In order to elucidate the function of human PIF1 helicase from biochemical level, we cloned human PIF1 gene by PCR from HeLa cell cDNA library. We co-transformed a pMStRNA1 plasmid encoding rare tRNA codons and a plasmid encoding molecular chaperon to greatly enhance the overexpression of human PIF1 protein. Finally we purified full-length PIF1 helicase by column chromatograph carried out at 4 degrees C using fast protein liquid chromatograph (FPLC) system. The human PIF1 protein was purified in enough quantity for detailed biochemical analysis. Biochemical assay showed that PIF1 had ATPase activity and helicase activity. The purification and biochemical properties analysis of human PIF1 helicase will allow us to understand how, at the molecular and mechanistic level, this conserved helicase operates in the cell.
DNA Helicases ; biosynthesis ; genetics ; metabolism ; HeLa Cells ; Humans ; RNA, Transfer ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism

OBJECTIVETo study the effect of fleroxacin (FLRX) on biological properties of Bloom (BLM) helicase catalytic core (BLM642-1290 helicase) in vitro and the molecular mechanism of interaction between the two molecules.

METHODSDNA-binding and unwinding activities of BLM642-1290 helicase were assayed by fluorescence polarization and gel retardation assay under conditions that the helicase was subjected to different concentrations of FLRX. Effect of FLRX on helicase ATPase activity was analyzed by phosphorus-free assay based on a colorimetric estimation of ATP hydrolysis-produced inorganic phosphate. Molecular mechanism of interaction between the two molecules was assayed by ultraviolet and fluorescence spectra.

RESULTSThe DNA unwinding and ATPase activities of BLM642-1290 helicase were inhibited whereas the DNA-binding activity was promoted in vitro. A BLM-FLRX complex was formed through one binding site, electrostatic and hydrophobic interaction force. Moreover, the intrinsic fluorescence of the helicase was quenched by FLRX as a result of non-radioactive energy transfer. The biological activity of helicase was affected by FLRX, which may be through an allosteric mechanism and stabilization of enzyme conformation in low helicase activity state, disruption of the coupling of ATP hydrolysis to unwinding, and blocking helicase translocation on DNA strands.

CONCLUSIONFLRX may affect the biological activities and conformation of BLM642-1290 helicase, and DNA helicase may be used as a promising drug target for some diseases.

DNA ; metabolism ; Fleroxacin ; pharmacology ; Nucleic Acid Synthesis Inhibitors ; pharmacology ; RecQ Helicases ; antagonists & inhibitors ; metabolism ; Spectrometry, Fluorescence ; Spectrophotometry, Ultraviolet

OBJECTIVE: To assess the value of m⁷G-lncRNAs in predicting the prognosis and microenvironment of colorectal cancer (CRC). METHODS: We screened m⁷G-lncRNAs from TCGA to construct an m⁷G-lncRNAs risk model using multivariate Cox analysis, which was validated using ROC and C-index curves. Calibration and nomogram were used to predict the prognosis of CRC patients. Point-bar charts and K-M survival curves were used to assess the correlation of risk scores with the patients' clinical staging and prognosis. CIBERSORT and ESTIMATE were used to explore the association between the tumor microenvironment and immune cell infiltration in patients in high and low risk groups and the correlation of risk scores with microsatellite instability, stem cell index and immune checkpoint expression. A protein-protein interaction network was constructed, and the key targets regulated by m⁷G-lncRNAs were identified and validated in paired samples of CRC and adjacent tissues by immunoblotting. RESULTS: We identified a total of 1722 m⁷G-lncRNAs from TCGA database, from which 12 lncRNAs were screened to construct the risk model. The AUCs of the risk model for predicting survival outcomes at 1, 3 and 5 years were 0.727, 0.747 and 0.794, respectively. The AUC of the nomogram for predicting prognosis was 0.794, and the predicted results were consistent with actual survival outcomes of the patients. The patients in the high-risk group showed more advanced tumor stages and a greater likelihood of high microsatellite instability than those in the low-risk group (P < 0.05). The tumor stemness index was negatively correlated with the risk score (r=-0.19; P=7.3e-05). Patients in the high-risk group had higher stromal cell scores (P=0.0028) and higher total scores (P=0.007) with lowered expressions of activated mast cells (r=-0.11; P=0.045) and resting CD4+ T cells (r=-0.14; P=0.01) and increased expressions of most immune checkpoints (P < 0.05). ATXN2 (P= 0.006) and G3BP1 (P=0.007) were identified as the key targets regulated by m⁷G-lncRNAs, and their expressions were both higher in CRC than in adjacent tissues. CONCLUSION The risk model based on 12 m⁷G-lncRNAs has important prognostic value for CRC and can reflect the microenvironment and the efficacy of immunotherapy in the patients.
Biomarkers, Tumor/metabolism* ; Colonic Neoplasms ; DNA Helicases/metabolism* ; Gene Expression Regulation, Neoplastic ; Humans ; Microsatellite Instability ; Poly-ADP-Ribose Binding Proteins/metabolism* ; Prognosis ; RNA Helicases/metabolism* ; RNA Recognition Motif Proteins/metabolism* ; RNA, Long Noncoding/metabolism* ; Tumor Microenvironment

RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination. However, the specific pathway(s) in which it is involved and the underlining mechanism(s) remain poorly understood. We took advantage of genetic tools in Drosophila to investigate how Drosophila RecQ5 (dRecQ5) functions in vivo in homologous recombination-mediated double strand break (DSB) repair. We generated null alleles of dRecQ5 using the targeted recombination technique. The mutant animals are homozygous viable, but with growth retardation during development. The mutants are sensitive to both exogenous DSB-inducing treatment, such as gamma-irradiation, and endogenously induced double strand breaks (DSBs) by I-Sce I endonuclease. In the absence of dRecQ5, single strand annealing (SSA)-mediated DSB repair is compromised with compensatory increases in either inter-homologous gene conversion, or non-homologous end joining (NHEJ) when inter-chromosomal homologous sequence is unavailable. Loss of function of dRecQ5 also leads to genome instability in loss of heterozygosity (LOH) assays. Together, our data demonstrate that dRecQ5 functions in SSA-mediated DSB repair to achieve its full efficiency and in suppression of LOH in Drosophila.
Animals ; DNA Repair ; genetics ; DNA, Single-Stranded ; genetics ; Drosophila Proteins ; genetics ; metabolism ; Drosophila melanogaster ; genetics ; metabolism ; Loss of Heterozygosity ; genetics ; RecQ Helicases ; genetics ; metabolism

The cranial neural crest plays a fundamental role in orofacial development and morphogenesis. Accordingly, mutations with impact on the cranial neural crest and its development lead to orofacial malformations such as cleft lip and palate. As a pluripotent and dynamic cell population, the cranial neural crest undergoes vast transcriptional and epigenomic alterations throughout the formation of facial structures pointing to an essential role of factors regulating chromatin state or transcription levels. Using CRISPR/Cas9-guided genome editing and conditional mutagenesis in the mouse, we here show that inactivation of Kat5 or Ep400 as the two essential enzymatic subunits of the Tip60/Ep400 chromatin remodeling complex severely affects carbohydrate and amino acid metabolism in cranial neural crest cells. The resulting decrease in protein synthesis, proliferation and survival leads to a drastic reduction of cranial neural crest cells early in fetal development and a loss of most facial structures in the absence of either protein. Following heterozygous loss of Kat5 in neural crest cells palatogenesis was impaired. These findings point to a decisive role of the Tip60/Ep400 chromatin remodeling complex in facial morphogenesis and lead us to conclude that the orofacial clefting observed in patients with heterozygous KAT5 missense mutations is at least in part due to disturbances in the cranial neural crest.
Animals ; Mice ; Chromatin Assembly and Disassembly ; Cleft Lip/genetics* ; Cleft Palate/genetics* ; DNA Helicases/metabolism* ; DNA-Binding Proteins ; Neural Crest/metabolism* ; Skull ; Transcription Factors/metabolism*

OBJECTIVETo study the effect of BRG1 and BRM, the catalytic subunits expressed by SWI/SNF, in benign and malignant prostatic tissues and to correlate the BRG1/BRM expression with the development and progression of prostatic cancer.

METHODSThe expression levels of the BRG1 and BRM proteins in benign and malignant prostatic tissues were studied using semi-quantitative immunohisto-chemistry. The results correlated with various clinical and pathologic parameters.

RESULTSThe average immuno-reactive score for BRG1 expression in prostatic cancer tissues was significantly higher than that in benign prostatic tissues (57+/-9.8 and 19+/-4.1, respectively, P = 0.000 17). The difference was more obvious in the high-grade cancer. On the other hand, BRM expression exhibited a heterogeneous pattern. The average immuno-reactive score for BRM expression was lower in cancer tissues than in benign tissues (112+/-17 and 151+/-19, respectively, P = 0.0047). BRG1 and BRM demonstrated a reciprocal expression pattern in benign and malignant tissues. The average immuno-reactive score for BRG1 expression was higher in the cancer cases with a larger tumor volume than in the cases with a smaller tumor volume (P = 0.0112).

CONCLUSIONSThe expression of BRG1 and BRM correlates with the development of prostatic cancer. Increased BRG1 expression may have certain implications in tumor progression.

DNA Helicases ; metabolism ; Disease Progression ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Nuclear Proteins ; metabolism ; Prostate ; metabolism ; pathology ; Prostatic Neoplasms ; metabolism ; pathology ; Transcription Factors ; metabolism ; Tumor Burden

OBJECTIVETo explore the relationship between SULF2 and WRN promoter methylation and chemosensitivity to irinotecan, and also the clinicopathological features in patients with gastric cancer.

METHODSThe chemosensitivity to irinotecan was tested by MTT assay. The methylation of SULF2 and WRN promoter in the fresh gastric cancer tissues was detected by methylation specific PCR. The differences of chemosensitivity and clinicopathological features of the methylation group were compared with that of the non-methylation group. The tumor growth in nude mice bearing human gastric cancer xenografts treated with CPT-11was also observed.

RESULTSThe methylation rates of SULF2 and WRN were 28.4% (29/102) and 23.5% (24/102), respectively. There were no significant association between promoter methylation and clinicopathological features of patients including age, gender, histologic type, lymphatic invasion, and TNM Stage. In all the 102 cases, there were 30 cases of irrinotecan-sensitive group, and 72 cases of the irrinotecan-resistant group. The SULF2 methylation rate was 46.7% (14/30)in the sensitive group, and 20.8% (15/72) in the resistant group (P = 0.008),The WRN methylation rate was 33.3% (10/30) in the sensitive group, and 19.4% (14/72) in the resistant group (P = 0.214). Gastric cancer tissues were more sensitive to irrinotecan when both the genes were methylated. The nude mice bearing human gastric cancer xenografts with SULF2 methylation were more sensitive to irrinotecan.

CONCLUSIONSThe detection of SULF2 and WRN promoter methylation may provide evidence for screening and targeting the most sensitive gastric cancer subpopulation suitable for personalized irrinotecan chemotherapy.

Antineoplastic Agents, Phytogenic ; pharmacology ; Camptothecin ; analogs & derivatives ; pharmacology ; DNA Methylation ; Exodeoxyribonucleases ; metabolism ; Humans ; Methylation ; Promoter Regions, Genetic ; RecQ Helicases ; metabolism ; Stomach Neoplasms ; metabolism ; Sulfotransferases ; metabolism ; Werner Syndrome Helicase

Objective: To investigate the clinicopathological features and differential diagnosis of olfactory carcinoma (OC). Methods: Twenty-one cases of sinonasal tumors, including those initially diagnosed as olfactory neuroblastoma (ONB) and those with uncertain diagnosis, were collected from the Department of Pathology, the First Affiliated Hospital of University of Science and Technology of China (Anhui Provincial Hospital) from January 2016 to August 2022, among which 3 cases were reclassified as OC. The clinicopathological features were investigated, and the remaining 18 cases were used as control. Results: Of the three OC patients, 2 were male and 1 was female, with an average age of 57 years ranging from 35 to 74 years. Microscopically, the tumor cells were arranged in solid, nested or lobulated patterns with occasional palisading around the solid nests. The stroma was highly vascular with focal neurofibrillary areas. There were prominent rosettes or pseudorosettes formation. The tumor cells were mainly ovoid to spindly with scant to moderate amount of cytoplasm, one or several small nucleoli, and fine chromatin content. Brisk mitotic figures were seen. In all 3 cases of OC, there were scanty atypical glands and some were ciliated. Immunohistochemically, at least one epithelial marker and neuroendocrine marker were diffusely expressed in the tumor. Some of the tumor cells were positive for p40 and p63, and the sustentacular cells showed the expression of S-100 protein. All cases tested were negative for NUT, CD99 and desmin, with intact expression of SMARCA4 (BRG1) and SMARCB1 (INI-1). Ki-67 proliferation index varied from 20% to 80%. Follow-up after 16-18 months showed no mortality with tumor recurrence from 1 patient after 16 months. Conclusion: OC is a rare sinonasal tumor with neuroepithelial differentiation, its histomorphology is diverse, and the combination of immunohistochemical markers is essential for appropriate diagnosis.
Humans ; Male ; Female ; Middle Aged ; Paranasal Sinus Neoplasms/chemistry* ; Biomarkers, Tumor/metabolism* ; Carcinoma/chemistry* ; Diagnosis, Differential ; S100 Proteins ; DNA Helicases/metabolism* ; Nuclear Proteins/metabolism* ; Transcription Factors/metabolism*

Chromodomain-helicase-DNA-binding protein 1 (CHD1) deletion is among the most common mutations in prostate cancer (PCa), but its role remains unclear. In this study, RNA sequencing was conducted in PCa cells after clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-based CHD1 knockout. Gene set enrichment analysis (GSEA) indicated upregulation of hypoxia-related pathways. A subsequent study confirmed that CHD1 deletion significantly upregulated hypoxia-inducible factor 1α (HIF1α) expression. Mechanistic investigation revealed that CHD1 deletion upregulated HIF1α by transcriptionally downregulating prolyl hydroxylase domain protein 2 (PHD2), a prolyl hydroxylase catalyzing the hydroxylation of HIF1α and thus promoting its degradation by the E3 ligase von Hippel-Lindau tumor suppressor (VHL). Functional analysis showed that CHD1 deletion promoted angiogenesis and glycolysis, possibly through HIF1α target genes. Taken together, these findings indicate that CHD1 deletion enhances HIF1α expression through PHD2 downregulation and therefore promotes angiogenesis and metabolic reprogramming in PCa.
Male ; Humans ; Von Hippel-Lindau Tumor Suppressor Protein/metabolism* ; DNA-Binding Proteins/metabolism* ; Prolyl Hydroxylases/metabolism* ; Hypoxia ; Prostatic Neoplasms/pathology* ; Glycolysis ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Cell Line, Tumor ; DNA Helicases/metabolism*

OBJECTIVETo investigate the expression of gene BRG1 in prostatic intraepithelial neoplasia and adenocarcinoma, and the relationship between gene BRG1 expression and the clinicopathological features of prostate carcinoma.

METHODSGene BRG1 expression was evaluated in 37 cases of human prostate carcinoma, 13 human prostatic intraepithelial neoplasia (PIN) and 14 human benign prostatic hyperplasia (BPH) by using immunohistochemistry (EnVision method) and tissue microarray.

RESULTSThe positive rates of BRG1 protein were 81.08% (30/37), 38.46% (5/13) and 14.28% (2/14) in prostate carcinoma, PIN and BPH, respectively, significantly higher in the first group than in the latter two (P < 0.05). There was no statistically significant difference in BRG1 gene expression either between PIN and BPH (P > 0.05) or between the groups of the moderate differentiation (the Gleason histologic grading: 5-7) and the lower one (the Gleason histologic grading: 8-10) (P > 0.05).

CONCLUSIONBRG1 may play an important role in the development of prostate carcinoma. Tissue microarray technology, with the advantages of high throughput, conciseness, rapidity, high efficiency, low cost, and nice reproducibility, has significant practical value and broad application prospects in pathology.

Aged ; Aged, 80 and over ; DNA Helicases ; biosynthesis ; Humans ; Immunohistochemistry ; Male ; Microchip Analytical Procedures ; Middle Aged ; Nuclear Proteins ; biosynthesis ; Prostatic Neoplasms ; metabolism ; pathology ; Reproducibility of Results ; Transcription Factors ; biosynthesis