Bacterial Proteins ; chemistry ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; genetics ; metabolism ; Gene Expression Regulation, Bacterial ; physiology ; Glutathione ; metabolism ; Listeria monocytogenes ; chemistry ; genetics ; metabolism ; Peptide Termination Factors ; chemistry ; genetics ; metabolism

OBJECTIVETo develop a method for enriching methylated DNA in clinical samples using mesocellular silica foams (MCFs) immobilized with methyl-CpG binding domain (MBD).

METHODSMCFs with ultra-large pore size were synthesized, functionalized and immobilized with GST-MBD.

RESULTSThe large cage-like pore structures of MCF materials was retained after functionalization and immobilization, with pore diameter of 55 nm, window size of 30 nm, and a high pore volume of 1.0 cm(3)/g. The loading amount of MBD was as high as 53 wt%. Immobilized MBD showed high binding activity and stability. In a binding buffer with salt concentrations ranging 500-550 mmol/L, the MCF-MBD can selectively enrich methylated DNA from the mixed DNA solution.

CONCLUSIONThe MCF-MBD method may offer a better choice for high-throughout DNA methylation screening, and has laid a foundation for clinical application, prenatal diagnosis and research on DNA methylation-related genetic diseases.

Animals ; CpG Islands ; DNA ; chemistry ; genetics ; metabolism ; DNA Methylation ; DNA-Binding Proteins ; chemistry ; Immobilized Proteins ; chemistry ; Protein Structure, Tertiary ; Rats ; Silicon Dioxide ; chemistry

DNA stable-isotope probing (DNA-SIP) is a recently developed method with which the incorporation of stable isotope from a labeled substrate is used to identify the function of microorganisms in the environment. The technique has now been used in conjunction with metagenomics to establish links between microbial identity and particular metabolic functions. The combination of DNA-SIP and metagenomics not only permits the detection of rare low-abundance species from metagenomic libraries but also facilitates the detection of novel enzymes and bioactive compounds. We summarize recent progress in SIP-metagenomic techniques and applications and discuss prospects for this combined approach in environmental microbiology and biotechnology.
Animals ; DNA ; genetics ; DNA Probes ; chemistry ; genetics ; metabolism ; DNA, Bacterial ; chemistry ; genetics ; metabolism ; Humans ; Isotope Labeling ; methods ; Metagenomics ; methods ; Molecular Probe Techniques ; Sequence Analysis, DNA ; methods

Eukaryotic cells contain numerous iron-requiring proteins such as iron-sulfur (Fe-S) cluster proteins, hemoproteins and ribonucleotide reductases (RNRs). These proteins utilize iron as a cofactor and perform key roles in DNA replication, DNA repair, metabolic catalysis, iron regulation and cell cycle progression. Disruption of iron homeostasis always impairs the functions of these iron-requiring proteins and is genetically associated with diseases characterized by DNA repair defects in mammals. Organisms have evolved multi-layered mechanisms to regulate iron balance to ensure genome stability and cell development. This review briefly provides current perspectives on iron homeostasis in yeast and mammals, and mainly summarizes the most recent understandings on iron-requiring protein functions involved in DNA stability maintenance and cell cycle control.
Animals ; Cell Cycle Checkpoints ; DNA ; metabolism ; DNA Repair ; DNA Replication ; Hemeproteins ; genetics ; metabolism ; Iron ; chemistry ; metabolism ; Iron-Sulfur Proteins ; genetics ; metabolism ; Ribonucleotide Reductases ; genetics ; metabolism ; Yeasts ; metabolism

8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a mutagenic adduct formed by reactive oxygen species. In Escherichia coli, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) removes this mutagenic adduct from DNA. In this report, we demonstrate base excision repair (BER) synthesis of DNA containing 8-oxo-G with Fpg in vitro. Fpg cut the oligonucleotide at the site of 8-oxo-G, producing one nucleotide gap with 3' and 5' phosphate termini. Next, 3' phosphatase(s) in the supernatant obtained by precipitating a crude extract of E. coli with 40% ammonium sulfate, removed the 3' phosphate group at the gap, thus exposing the 3' hydroxyl group to prime DNA synthesis. DNA polymerase and DNA ligase then completed the repair. These results indicate the biological significance of the glycosylase and apurinic/ apyrimidinic (AP) lyase activities of Fpg, in concert with 3' phosphatase(s) to create an appropriately gapped substrate for efficient BER synthesis of DNA containing 8-oxo-G.
DNA Glycosylases/metabolism ; *DNA Repair ; DNA, Bacterial/*chemistry/*metabolism ; DNA-Formamidopyrimidine Glycosylase/metabolism ; Escherichia coli/*enzymology/*genetics ; Guanine/*analogs & derivatives/*metabolism

Large doses of acetaminophen (APAP) could cause oxidative stress and tissue damage through production of reactive oxygen/nitrogen (ROS/RNS) species and quinone metabolites of APAP. Although ROS/RNS are known to modify DNA, the effect of APAP on DNA modifications has not been studied systematically. In this study, we investigate whether large doses of APAP can modify the nuclear DNA in C6 glioma cells used as a model system, because these cells contain cytochrome P450-related enzymes responsible for APAP metabolism and subsequent toxicity (Geng and Strobel, 1995). Our results revealed that APAP produced ROS and significantly elevated the 8-oxo- deoxyguanosine (8-oxodG) levels in the nucleus of C6 glioma cells in a time and concentration dependent manner. APAP significantly reduced the 8- oxodG incision activity in the nucleus by decreasing the activity and content of a DNA repair enzyme, Ogg1. These results indicate that APAP in large doses can increase the 8-oxodG level partly through significant reduction of Ogg1 DNA repair enzyme.
Acetaminophen/*metabolism ; Analgesics, Non-Narcotic/*metabolism ; Animals ; Cell Line, Tumor ; DNA/metabolism ; DNA Damage ; DNA Glycosylases/*metabolism ; DNA Repair ; Deoxyguanosine/chemistry/*metabolism ; Glioma/*metabolism ; Glutathione/metabolism ; Humans ; Rats ; Reactive Nitrogen Species/metabolism ; Reactive Oxygen Species/metabolism

SWI1 is a member of a new class of tumor DNA-binding proteins named as the AT-rich interaction domain family (ARID), and considered to bind with AT base pairs specifically. Genomic and functional data support ARID1A as a tumor suppressor because ARID1A/BAF250a (SWI1) subunit of the SWI/SNF chromatin-remodeling complex has emerged as recurrently mutated in a broad array of tumor types. But the crystal structure of SWI1 has not been solved as yet. Using docking and molecular dynamics, we predicted the DNA interaction pattern of human SWI1 ARID and made comparisons with the other two representative ARID family members, human Mrf-2 ARID and Drosophila Dri ARID. Dynamic results revealed that the N-terminal and loop L1 of SWI1 ARID bound with the DNA major groove, while the loop L2 and helix H6 bound with the minor groove. Moreover, it was found that SWI1 ARID bound with DNA apparently in a sequence-nonspecific manner. It was concluded that SWI1 ARID can form stable complex with sequence-nonspecific DNA segment comparing to Mrf-2 ARID/DNA and Dri ARID/DNA sequence-specific complexes.
Binding Sites ; DNA ; chemistry ; metabolism ; DNA-Binding Proteins ; chemistry ; metabolism ; Drosophila Proteins ; chemistry ; Homeodomain Proteins ; chemistry ; Humans ; Models, Molecular ; Molecular Docking Simulation ; Molecular Dynamics Simulation ; Nuclear Proteins ; chemistry ; Protein Structure, Tertiary ; Transcription Factors ; chemistry ; metabolism

Base Sequence ; DNA ; chemistry ; DNA-Directed DNA Polymerase ; metabolism ; Fluorescent Dyes ; chemistry ; Humans ; Molecular Sequence Data ; Nanostructures ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA ; methods

Animals ; DNA Methylation ; DNA-Cytosine Methylases ; chemistry ; metabolism ; physiology ; Embryonic Development ; physiology ; Humans ; Mice ; Site-Specific DNA-Methyltransferase (Adenine-Specific) ; chemistry ; metabolism ; physiology

Zinc-finger proteins have been widely studied due to their highly conserved structures and DNA-binding specificity of zinc-finger domains. However, researches on the zinc-finger proteins from microorganisms, especially those from prokaryotes, are still very limited. This review focuses on the latest progress on microbial zinc-finger proteins, especially those from prokaryotes and the application of artificial zinc-finger proteins in the breeding of robust strains. Artificial zinc-finger proteins with transcriptional activation or repression domain can regulate the global gene transcription of microbial cells to acquire improved phenotypes, such as stress tolerance to heat, ethanol, butanol, and osmotic pressure. Using the zinc-finger domain as DNA scaffold in the construction of enzymatic system can enhance the catalytic efficiency and subsequently the production of specific metabolites. Currently, zinc-finger domains used in the construction of artificial transcription factor are usually isolated from mammalian cells. In the near future, novel transcription factors can be designed for strain development based on the natural zinc-finger domains from different microbes, which may be used to regulate the global gene expression of microbial cells more efficiently.
Bacteria ; metabolism ; DNA ; chemistry ; Protein Engineering ; Transcription Factors ; chemistry ; Transcriptional Activation ; Zinc Fingers