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

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

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

Transcription activator-like (TAL) effectors specifically bind to double stranded (ds) DNA through a central domain of tandem repeats. Each TAL effector (TALE) repeat comprises 33-35 amino acids and recognizes one specific DNA base through a highly variable residue at a fixed position in the repeat. Structural studies have revealed the molecular basis of DNA recognition by TALE repeats. Examination of the overall structure reveals that the basic building block of TALE protein, namely a helical hairpin, is one-helix shifted from the previously defined TALE motif. Here we wish to suggest a structure-based re-demarcation of the TALE repeat which starts with the residues that bind to the DNA backbone phosphate and concludes with the base-recognition hyper-variable residue. This new numbering system is consistent with the α-solenoid superfamily to which TALE belongs, and reflects the structural integrity of TAL effectors. In addition, it confers integral number of TALE repeats that matches the number of bound DNA bases. We then present fifteen crystal structures of engineered dHax3 variants in complex with target DNA molecules, which elucidate the structural basis for the recognition of bases adenine (A) and guanine (G) by reported or uncharacterized TALE codes. Finally, we analyzed the sequence-structure correlation of the amino acid residues within a TALE repeat. The structural analyses reported here may advance the mechanistic understanding of TALE proteins and facilitate the design of TALEN with improved affinity and specificity.
Adenine ; chemistry ; metabolism ; Amino Acid Sequence ; Binding Sites ; DNA ; chemistry ; metabolism ; DNA-Binding Proteins ; chemistry ; metabolism ; Guanine ; chemistry ; metabolism ; Molecular Dynamics Simulation ; Molecular Sequence Data ; Protein Binding ; Protein Structure, Secondary ; Protein Structure, Tertiary

OBJECTIVEExpress and purify four single-stranded DNA-binding (SSB) proteins, and evaluate the binding of SSB proteins on HCV RNA.

METHODSThe expression plasmids of four SSB proteins were conducted, termed TTH, SSOB, KOD and BL21, respectively. The BL21 (DE3) was transformed by the expression plasmid of TTH, Transetta (DE3) were transformed by the expression plasmid of SSOB, KOD and BL21, then protein expression was induced with IPTG, the expression products were analysised by SDS-PAGE. To evaluate the binding of SSB on HCV RNA, RNA-SSB protein complexes were applied to a 1.2% TAE agarose gel.

RESULTSSuitable competent cells were transformed with the expression plasmids, induced by IPTG. SSB proteins were purified by affinity chromatography, to visualize their purity all SSB proteins were applied to SDS-PAGE analysis. All four proteins showed single clear bands. We have successfully obtained the SSB protein expression plasmid, expressed and purified SSB protein. TAE agarose gel electrophoresis was used to confirm SSB protein-RNA binding activity. The each of SSB-RNA complex migrated more slowly than the sole RNA, which suggested SSB protein could specifically bind to RNA.

CONCLUSIONSWe have expressed and purified four SSB proteins, and for the first time found that SSB protein can bind HCV RNA. Our results may provide a basis for future studies of the novel functions of SSB proteins on RNA.

DNA, Single-Stranded ; genetics ; metabolism ; DNA-Binding Proteins ; chemistry ; isolation & purification ; metabolism ; Hepacivirus ; Hepatitis C ; metabolism ; virology ; Humans ; Molecular Weight ; Protein Binding ; RNA, Viral ; genetics ; metabolism

OBJECTIVETo establish a new non-radioactive method for electrophoretic mobility shift assay (EMSA) to investigate the binding between glucocorticoid induced leucine zipper (GILZ) and peroxisome proliferator-activated receptor-gamma 2 (PPARgamma2) promoter oligonucleotides.

METHODSGILZ protein prepared by prokaryotic expression was linked to PPARgamma2 promoter oligonucleotides end-labeled with IRDye 800 infrared dye. The DNA-protein complex was separated with non-denatured polyacrylamide gel and scanned with the Odyssey. Infrared Imaging System.

RESULTSOne lane of DNA-protein complex was clearly presented, and the signal intensity increased along with the increment of the protein load.

CONCLUSIONThis infrared imaging system can be used for EMSA for detecting the DNA-protein complex with high sensitivity efficiency and allows easy operation.

Binding Sites ; DNA ; chemistry ; DNA-Binding Proteins ; chemistry ; metabolism ; Electrophoretic Mobility Shift Assay ; instrumentation ; methods ; Fluorescent Dyes ; chemistry ; Gene Expression Regulation ; Humans ; Infrared Rays ; Protein Binding ; Protein Interaction Domains and Motifs ; physiology ; Proteins ; 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

Protein folding, stability, and function are usually influenced by pH. And free energy plays a fundamental role in analysis of such pH-dependent properties. Electrostatics-based theoretical framework using dielectric solvent continuum model and solving Poisson-Boltzmann equation numerically has been shown to be very successful in understanding the pH-dependent properties. However, in this approach the exact computation of pH-dependent free energy becomes impractical for proteins possessing more than several tens of ionizable sites (e.g. > 30), because exact evaluation of the partition function requires a summation over a vast number of possible protonation microstates. Here we present a method which computes the free energy using the average energy and the protonation probabilities of ionizable sites obtained by the well-established Monte Carlo sampling procedure. The key feature is to calculate the entropy by using the protonation probabilities. We used this method to examine a well-studied protein (lysozyme) and produced results which agree very well with the exact calculations. Applications to the optimum pH of maximal stability of proteins and protein-DNA interactions have also resulted in good agreement with experimental data. These examples recommend our method for application to the elucidation of the pH-dependent properties of proteins.
Cathepsin B ; chemistry ; metabolism ; DNA ; metabolism ; Hydrogen-Ion Concentration ; Molecular Dynamics Simulation ; Monte Carlo Method ; Muramidase ; chemistry ; metabolism ; Probability ; Protein Binding ; Proteins ; chemistry ; metabolism ; Protons ; Thermodynamics

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

By using an RAD peptide display system derived from the ATPase domain of recombinase RadA of Pyrococcus furiosus, an anti-hCG antibody-like molecule was prepared by grafting an hCG-binding peptide to the RAD scaffold. After linking to sfGFP gene, a gene of hCG peptide-grafted RAD was synthesized and cloned into a bacterial expression vector (pET30a-RAD/hCGBP-sfGFP). The vector was transformed into Escherichia coli, and expression of the fusion protein was induced. After isolation and purification of the fusion protein, its binding affinity and specificity to hCG were determined by using a process of immunoabsorption followed by GFP fluorescence measurement. A comparison of hCG-binding activity with a similarly grafted single-domain antibody based on a universal scaffold was performed. The measurement of hCG-binding affinity and specificity revealed that the grafted RAD has an optimally high binding affinity and specificity to hCG, which are better than the grafted single-domain antibody. Moreover, the affinity and specificity of grafted RAD molecule are comparable to those of a commercial monoclonal antibody. In addition, the hCG-binding peptide-grafted RAD molecule has a relatively high biochemical stability, making it a good substitute for antibody with potential application.
Antibodies, Monoclonal ; chemistry ; isolation & purification ; metabolism ; Antibody Specificity ; DNA-Binding Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; Escherichia coli Proteins ; metabolism ; Humans ; Peptides ; Recombinant Fusion Proteins ; genetics ; metabolism