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

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

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

Genome duplication in E. coli is carried out by DNA polymerase III, an enzyme complex consisting of ten subunits. Investigations of the biochemical and structural properties of DNA polymerase III require the expression and purification of subunits including α, ge, θ, γ, δ', δ, and β separately followed by in vitro reconstitution of the pol III core and clamp loader. Here we propose a new method for expressing and purifying DNA polymerase III components by utilizing a protein co-expression strategy. Our results show that the subunits of the pol III core and those of the clamp loader can be coexpressed and purified based on inherent interactions between the subunits. The resulting pol III core, clamp loader and sliding clamp can be reconstituted effectively to perform DNA polymerization. Our strategy considerably simplifies the expression and purification of DNA polymerase III and provides a feasible and convenient method for exploring other multi-subunit systems.
Cloning, Molecular ; DNA Polymerase III ; chemistry ; genetics ; metabolism ; DNA Replication ; DNA, Bacterial ; biosynthesis ; genetics ; Escherichia coli ; enzymology ; genetics ; Plasmids ; metabolism ; Polymerization ; Protein Engineering ; methods ; Protein Subunits ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism

In this new era of the genome, the complete sequences of various organisms (from the simplest to the most complex such as human) are now available, which provides new opportunities to study biology and to develop therapeutic strategies. But the paucity of research tools that manipulate specific genes in vivo represents a major limitation of functional genomic studies. In nature, the expression of genes is regulated at the transcriptional level primarily by proteins that bind to nucleic acids. Many of these proteins, which are termed transcription factors, are typically consist of two essential yet separable modules: DNA-binding domain (DBD) and effector domain (ED). Attempts to control the gene expression by artificial transcription factors are based on the application of this rule. Among the many naturally occurring DNA-binding domains, the Cys2-His2 zinc-finger domain has demonstrated the greatest potential for the design of novel sequence-specific DNA-binding proteins. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. Ideally, these artificial DNA binding proteins could be designed to specifically target and regulate one single gene within a genome as complex as that found in human. Such proteins would be powerful tools in basic and applied research.
DNA ; chemistry ; genetics ; metabolism ; DNA-Binding Proteins ; metabolism ; Gene Expression Regulation ; Humans ; Transcription Factors ; chemistry ; genetics ; metabolism ; Zinc Fingers ; genetics ; physiology

In addition to DNA repair pathways, cells utilize translesion DNA synthesis (TLS) to bypass DNA lesions during replication. During TLS, Y-family DNA polymerase (Polη, Polκ, Polı and Rev1) inserts specific nucleotide opposite preferred DNA lesions, and then Polζ consisting of two subunits, Rev3 and Rev7, carries out primer extension. Here, we report the complex structures of Rev3-Rev7-Rev1(CTD) and Rev3-Rev7-Rev1(CTD)-Polκ(RIR). These two structures demonstrate that Rev1(CTD) contains separate binding sites for Polκ and Rev7. Our BIAcore experiments provide additional support for the notion that the interaction between Rev3 and Rev7 increases the affinity of Rev7 and Rev1. We also verified through FRET experiment that Rev1, Rev3, Rev7 and Polκ form a stable quaternary complex in vivo, thereby suggesting an efficient switching mechanism where the "inserter" polymerase can be immediately replaced by an "extender" polymerase within the same quaternary complex.
Binding Sites ; Crystallography, X-Ray ; DNA Repair ; DNA-Binding Proteins ; chemistry ; genetics ; metabolism ; DNA-Directed DNA Polymerase ; chemistry ; genetics ; metabolism ; Fluorescence Resonance Energy Transfer ; Humans ; Mad2 Proteins ; Nuclear Proteins ; chemistry ; genetics ; metabolism ; Nucleotidyltransferases ; chemistry ; genetics ; metabolism ; Protein Binding ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Proteins ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

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

OBJECTIVETo study the ethylene responsive element binding protein genes of Salvia miltiorrhiza through bioinformatics and characterization of its tissue expression in regenerated plantlets.

METHODThe ethylene responsive element binding protein genes were obtained by cDNA microarray analyze. BLAST was used for alignment, ORF finder software was used to find open reading frame, Prosite database was used to analyze the protein. Semi-quantitative RT- PCR method was used to detect the gene expression level.

RESULTOne ethylene responsive element binding protein was obtained, named as SmERF. SmERF had an open reading frame of 699 bp with 5'-URT 87 bp and 3'-URT 166 bp. The putative protein SmERF contains a highly conserved ERF/AP2 domain. Semiquantitative RT- PCR illustrated that SmERF was expressed in all tissues such as root, stem and leaf in regenerated shoots, while the expression level was higher in root than in stem and leaf.

CONCLUSIONIt was the first time to obtain ERF gene in S. miltiorrhiza and set a good foundation for its further functional study.

DNA-Binding Proteins ; chemistry ; genetics ; metabolism ; Gene Expression ; Genomics ; Open Reading Frames ; Plant Proteins ; chemistry ; genetics ; metabolism ; Protein Structure, Tertiary ; Salvia ; chemistry ; genetics ; metabolism ; Untranslated Regions

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

The preparation and cell transfection of chitosan-DNA nanoparticles were studied. The TFPI (tissue factor pathway inhibitor) or EGFP (enhanced green fluorescent protein) plasmid DNA was encapsulated with chitosan to form gene nanoparticles. The results with TEM showed that the nanoparticles were of sphere shape. The mean diameter of the nanoparticles was 149 nm and the diameter ranged from 80-250 nm, which were measured by the photo related spectrometry (PCS). The encapsulation efficiency of DNA was 96% +/- 1.38% and the DNA content in the nanoparticles was 37% +/- 3.0%. The encapsulated DNA could be protected from the degradation by DNase I. The transfection efficiency of chitosan nanoparticles were about equivalent to that of the LipofectAMINETM reagent. Our results also showed that chitosan nanoparticles were nontoxic to cultured cells.
Cells, Cultured ; Chitosan ; chemistry ; DNA ; chemistry ; genetics ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Lipoproteins ; genetics ; Muscle, Smooth, Vascular ; chemistry ; metabolism ; Nanoparticles ; chemistry ; Plasmids ; genetics ; Transfection