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

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

Atomic force microscopy (AFM) has been applied in many biological investigations in recent years, and this review focuses on the application of AFM in DNA-protein interactions. AFM images of static DNA-protein complexes, in air and in liquid, can be used to obtain quantitative and qualitative information on the structure of different complexes. And dynamic AFM images of DNA-protein complexation in real time under liquid conditions will help to understand biological processes and mechanisms at single molecule level. In addition, the measurement of intermolecular forces between biomolecules also provides new opportunities for studying mechanical properties of biomolecules and the interactions in their native environment. AFM has revealed many mechanisms of gene regulation, and will play a more and more important role in life science research.
DNA ; chemistry ; ultrastructure ; Humans ; Microscopy, Atomic Force ; methods ; Protein Binding ; Protein Interaction Mapping ; Proteins ; chemistry ; ultrastructure

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

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

Mixed linked leukemia 4 (MLL4) is a specific methyltransferase of histone 3 position lysine 4 (H3K4). It is also one of the important members of COMPASS/Set1-like protein complex. Both MLL4 protein itself and its mediated H3K4 methylation modification can cause changes in chromatin structure and function, thus regulating gene transcription and expression. With the studies of MLL4 protein in recent years, the roles of MLL4 gene, MLL4 protein and protein complex in the development of tissues and organs, tumor diseases and other physiological and pathophysiological processes have been gradually revealed. In this paper, the research progress of MLL4 gene, MLL4 protein characteristics, biological function and its effect on disease were reviewed, in order to further understand the effect of histone methyltransferase on gene expression regulation, as well as its non-enzyme dependent function. This paper may provide new ideas for the prevention, diagnosis and treatment of related diseases.
DNA-Binding Proteins ; physiology ; Histone-Lysine N-Methyltransferase ; physiology ; Histones ; chemistry ; Humans ; Methylation

As an important telomere binding protein, TPP1 protects the ends of telomeres and maintains the stability and integrity of its structure and function by interacting with other five essential core proteins (POT1, TRF1, TRF2, TIN2, and RAP1) to form a complex called Shelterin. Recently, researchers have discovered that TPP1 participates in protection of telomeres and regulation of telomerase activity. The relationship between TPP1 and tumorigenesis, tumor progression and treatment has also been investigated. This paper reviews the latest findings of TPP1 regarding to its structure, function and interaction with other proteins involved in tumorigenesis.
Chromosomal Instability ; DNA Damage ; Humans ; Neoplasms ; genetics ; Telomere ; Telomere-Binding Proteins ; chemistry ; physiology

High selectivity provided by biomolecules such as antibodies and enzymes has been exploited during the last two decades for development of biosensors. Of particular importance are efficient immobilization methods for biomolecules in order to preserve their biological activities. In this study, we have evaluated immobilization strategies for an anti-DNA antibody on a self-assembled monolayer of omega-functionalized thiols. The antibody was immobilized via peptide bond formation between the primary amines in the antibody and the carboxyl groups on the self-assembled monolayer. The peptide bond coupling was achieved by activating COOH groups on the surface through N-Hydroxysuccimide (NHS)-ester formation, followed by acylation of NH2 group in the antibody. DNA binding activity of the immobilized antibody was examined by counting beta emission from 35S-labeled DNA.
Antibodies, Antinuclear* ; DNA/immunology ; DNA/analysis* ; DNA-Binding Proteins/chemistry ; Gold ; Membranes, Artificial ; Polymerase Chain Reaction ; Polyvinyls/chemistry ; Radioimmunoassay/methods* ; Thioctic Acid/chemistry

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

Pulldown assay is an in vitro method for studies of protein-protein interactions, in which tagged proteins are usually expressed as the bait to enrich other proteins that could bind to them. In this technology, the GST tag is broadest used for its modest size and hydrophilic property. In most cases, the GST tag could increase the hydrophility of the fusion protein and help to avoid the formation of inclusion bodies. However, in the other few cases, the target protein may be strongly hydrophobic or have complicated structures that were hard to fold and assemble in correct conformations without champerons, and even the existence of GST tag could not make them soluble. These proteins were always expressed as inclusion bodies and had no functions. LMO2 was a small molecular weight and insoluble protein, in this study, GST system and MBP system were used to express GST-LMO2 and MBP-LMO2 fusion proteins, respectively. We found that GST-LMO2 fusion protein was expressed as inclusion bodies whereas MBP-LMO2 fusion protein was expressed in soluble form. Moreover, the production rate of MBP-LMO2 was also much higher than GST-LMO2. Then MBP-LMO2 fusion proteins and renatured GST-LMO2 fusion proteins were used as bait in pulldown assay to study the interaction between LMO2 and endogenous GATA1 in K562 cells. Western blot analyses showed that both of these proteins could bind to endogenous GATA1 in K562 cells, but recovered GATA1 protein by MBP-LMO2 fusion protein was much more than GST-LMO2 fusion protein. These results suggest that using of MBP system is a helpful attempt in the case of studying small molecular weight, strong hydrophobic proteins.
Adaptor Proteins, Signal Transducing ; Carrier Proteins ; chemistry ; Chemical Precipitation ; DNA-Binding Proteins ; chemistry ; GATA1 Transcription Factor ; chemistry ; Genetic Vectors ; Glutathione Transferase ; chemistry ; Humans ; K562 Cells ; LIM Domain Proteins ; Maltose-Binding Proteins ; Metalloproteins ; chemistry ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Renaturation ; Proto-Oncogene Proteins ; chemistry ; Recombinant Fusion Proteins ; genetics ; metabolism