'Calling Cards' method for high-throughput identification of targets of yeast DNA-binding proteins

We present a protocol for a novel method for identifying the targets of DNA-binding proteins in the genome of the yeast Saccharomyces cerevisiae. This is accomplished by engineering a DNA-binding protein so that it leaves behind in the genome a permanent mark -- a 'calling card' -- that provides a record of that protein's visit to that region of the genome. The calling card is the yeast Ty5 retrotransposon, whose integrase interacts with the Sir4 protein. If Sir4 is fused to a DNA-binding protein, it recruits the Ty5 integrase, which directs insertion of a Ty5 calling card into the genome. The calling card along with the flanking genomic DNA is harvested by inverse PCR and its genomic location is determined by hybridization of the product to a DNA microarray. This method provides a straightforward alternative to the 'ChIP-chip' method for determining the targets of DNA-binding proteins. This protocol takes approximately 2 weeks to complete.     

DNA-binding proteins in cells and membrane blebs of Neisseria gonorrhoeae.

Naturally elaborated membrane bleb fractions BI and BII of Neisseria gonorrhoeae contain both linear and circular DNAs. Because little is known about the interactions between DNA and blebs, studies were initiated to identify specific proteins that bind DNA in elaborated membrane blebs. Western immunoblots of whole-cell and bleb proteins from transformation-competent and DNA-uptake-deficient (dud) mutants were probed with single- or double-stranded gonococcal DNA, pBR322, or synthetic DNA oligomers containing intact or altered gonococcal transformation uptake sequences. The specificity and sensitivity of a nonradioactive DNA-binding protein assay was evaluated, and the assay was used to visualize DNA-protein complexes on the blots. The complexes were then characterized by molecular mass, DNA-binding specificity, and expression in bleb fractions. The assay effectively detected blotted DNA-binding proteins. At least 17 gonococcal DNA-binding proteins were identified; unique subsets occurred in BI and BII. Certain DNA-binding proteins had varied affinities for single- and double-stranded DNA, and the intact transformation uptake sequence competitively displaced the altered sequence from a BI protein at 11 kilodaltons (kDa). A dud mutant, strain FA660, lacked DNA-binding activity at the 11-kDa protein in BI. The segregation of DNA-binding proteins within BI and BII correlates with their distinct protein profiles and suggests that these vesicles may play different roles. Although the DNA-binding proteins expressed in BII may influence the nuclease-resistant export of plasmids within BII vesicles, the BI 11-kDa protein may bind transforming DNA.     

Genome-wide survey of DNA-binding proteins in Arabidopsis thaliana: analysis of distribution and functions

The interaction of proteins with their respective DNA targets is known to control many high-fidelity cellular processes. Performing a comprehensive survey of the sequenced genomes for DNA-binding proteins (DBPs) will help in understanding their distribution and the associated functions in a particular genome. Availability of fully sequenced genome of Arabidopsis thaliana enables the review of distribution of DBPs in this model plant genome. We used profiles of both structure and sequence-based DNA-binding families, derived from PDB and PFam databases, to perform the survey. This resulted in 4471 proteins, identified as DNA-binding in Arabidopsis genome, which are distributed across 300 different PFam families. Apart from several plant-specific DNA-binding families, certain RING fingers and leucine zippers also had high representation. Our search protocol helped to assign DNA-binding property to several proteins that were previously marked as unknown, putative or hypothetical in function. The distribution of Arabidopsis genes having a role in plant DNA repair were particularly studied and noted for their functional mapping. The functions observed to be overrepresented in the plant genome harbour DNA-3-methyladenine glycosylase activity, alkylbase DNA N-glycosylase activity and DNA-(apurinic or apyrimidinic site) lyase activity, suggesting their role in specialized functions such as gene regulation and DNA repair.     

Heterogeneity of nuclear proteins from HeLa cells specifically binding to the Alu sequence

Nuclear proteins from HeLa cells specifically binding to the Alu-repeat cloned in the plasmid Blur8 have been studied. 0.35 M nuclear extract proteins have been separated on DEAE-cellulose. The presence of DNA-binding proteins has been found in all fractions by the technique of DNA-binding on nitrocellulose filters. The labelled restricted DNA of the plasmid Blur8 was incubated with the proteins of different fractions with the subsequent identification of specific Alu-protein complexes in polyacrylamide gel at low ionic strength. At least two proteins have been found to have the different affinity to Alu-repeat. Various functions of Alu-repeats and the possibility of their participation in the initiation of DNA replication are discussed.     

Replication and recombination of herpes simplex virus DNA

Replication of herpes simplex virus takes place in the cell nucleus and is carried out by a replisome composed of six viral proteins: the UL30-UL42 DNA polymerase, the UL5-UL8-UL52 helicase-primase, and the UL29 single-stranded DNA-binding protein ICP8. The replisome is loaded on origins of replication by the UL9 initiator origin-binding protein. Virus replication is intimately coupled to recombination and repair, often performed by cellular proteins. Here, we review new significant developments: the three-dimensional structures for the DNA polymerase, the polymerase accessory factor, and the single-stranded DNA-binding protein; the reconstitution of a functional replisome in vitro; the elucidation of the mechanism for activation of origins of DNA replication; the identification of cellular proteins actively involved in or responding to viral DNA replication; and the elucidation of requirements for formation of replication foci in the nucleus and effects on protein localization.     

Detection of DNA-protein binding in western blots by phosphorus-labeled and biotinylated DNA probes

Eukaryotic DNA-binding proteins can be detected by a filter binding assay combining protein blotting on nitrocellulose, incubation with DNA by filtration, and the application of radioactively or nonradioactively labeled DNA probes. Basic nuclear and non-nuclear standard proteins are assayed in dot blots as well as in Western blots from sodium dodecyl sulfate gels. The DNA-binding ability of fractionated proteins is compared employing two different blotting techniques, conventional electro-transfer and protein-renaturating capillary transfer. Biotinylated DNA probes exhibit high sensitivity and a distinct discrimination of detection signals corresponding only to defined DNA-binding proteins. In contrast, phosphorus-labeled DNA probes show higher sensitivity, but less effective resolving power, especially for bands localized close to each other. Using the DNA-incubation procedure described, biotinylated DNA probes are preferable to radioactively-labeled probes for screening DNA-binding proteins in complex protein fractions.     

Novel DNA binding proteins highly specific to UV-damaged DNA sequences from embryos of Drosophila melanogaster.

Three new proteins which selectively bind to UV-damaged DNA were identified and purified to near homogeneity from UV-irradiated Drosophila melanogaster embryos through several column chromatographies. These proteins, tentatively designated as D-DDB P1, P2 and P3, can be identified as different complex bands in a gel shift assay by using UV-irradiated TC-31 probe DNA. Analysis of the purified D-DDB P1 fraction by native or SDS-polyacrylamide gel electrophoresis and FPLC-Superose 6 gel filtration demonstrated that it is a monomer protein which is a 30 kDa polypeptide. The D-DDB P2 protein is a monopolypeptide with a molecular mass of 14 kDa. Both D-DDB P1 and P2 highly prefer binding to UV-irradiated DNA, and have almost no affinity for non-irradiated DNA. Gel shift assays with either UV-irradiated DNA probes demonstrated that D-DDB P1 may show a preference for binding to (6-4) photoproducts, while D-DDB P2 may prefer binding to pyrimidine dimers. Both these proteins require magnesium ions for binding. D-DDB P1 is an ATP-preferent protein. These findings are discussed in relation to two recently described [Todo and Ryo (1991) Mutat. Res., 273, 85-93; Todo et al. (1993) Nature, 361, 371-374] DNA-binding factors from Drosophila cell extracts. A possible role for these DNA-binding proteins in lesion recognition and DNA-binding proteins in lesion recognition and DNA repair of UV-induced photo-products is discussed.     

DNA mimicry by proteins and the control of enzymatic activity on DNA

Cells are unable to perform any function on their DNA in the absence of proteins, and it is of vital importance that these proteins only perform their function at appropriate times during the cell cycle. Thus, DNA-binding proteins are always controlled by a wide range of other factors, primarily other proteins. These controlling factors usually block access of the protein to the DNA, often operating by simple competitive inhibition. However, it has recently been demonstrated that DNA-binding proteins can be controlled by the direct binding of the control protein to the DNA-binding site on the first protein. The structures of these control proteins have revealed that they mimic the structure and electrostatics of DNA. This review highlights the roles of DNA mimics in the control of DNA-binding proteins, suggests other possible candidate proteins using DNA mimicry, and puts forward a range of potential uses of DNA mimics.     

DNA-binding proteins of mammalian mitochondria

Acid-soluble proteins were isolated from liver and spleen mitochondria and their ability to form complexes with DNA was investigated. According to electrophoresis data, acid-soluble proteins include about 20 polypeptides ranging in the molecular mass from 10 to 120 kDa. It was found that acid-soluble proteins form stable DNA-protein complexes at a physiological NaCl concentration. Different polypeptides possess different degrees of DNA affinity. There is no significant difference between DNA-binding proteins of mitochondria from liver and those from spleen as to their ability to form complexes with mtDNA and nDNA. In the presence of 5 microg of DNA most polypeptides were bound to DNA, and further increase in DNA amount affected little the binding of proteins to DNA. There was no distinct difference in DNA-protein complex formation of liver mitochondrial acid-soluble proteins with nDNA or mtDNA. Also, it was detected that with these mitochondrial acid-soluble proteins, proteases that specifically cleave these proteins are associated. It was shown for the first time that these proteases are activated by DNA. DNA-binding proteins including DNA-activated mitochondrial proteases are likely to participate in the regulation of the structural organization and functional activity of mitochondrial DNA.     

High Free-Energy Barrier of 1D Diffusion Along DNA by Architectural DNA-Binding Proteins

Architectural DNA-binding proteins function to regulate diverse DNA reactions and have the defining property of significantly changing DNA conformation. Although the 1D movement along DNA by other types of DNA-binding proteins has been visualized, the mobility of architectural DNA-binding proteins on DNA remains unknown. Here, we applied single-molecule fluorescence imaging on arrays of extended DNA molecules to probe the binding dynamics of three structurally distinct architectural DNA-binding proteins: Nhp6A, HU, and Fis. Each of these proteins was observed to move along DNA, and the salt concentration independence of the 1D diffusion implies sliding with continuous contact to DNA. Nhp6A and HU exhibit a single sliding mode, whereas Fis exhibits two sliding modes. Based on comparison of the diffusion coefficients and sizes of many DNA binding proteins, the architectural proteins are categorized into a new group distinguished by an unusually high free-energy barrier for 1D diffusion. The higher free-energy barrier for 1D diffusion by architectural proteins can be attributed to the large DNA conformational changes that accompany binding and impede rotation-coupled movement along the DNA grooves.     

Use of DNA-protein interaction to isolate specific genomic DNA sequences.

We describe a simple and rapid method for the isolation of specific genomic DNA sequences recognized by DNA-binding proteins. This procedure consists of four steps: (1) restriction enzyme digestion and size fractionation of genomic DNA; (2) DNA--protein binding using the gel mobility-shift assay; (3) ligation of isolated DNA fragments followed by transformation of Escherichia coli; and (4) screening of recombinant clones for inserts containing specific DNA--protein binding sequences. We have used this protocol to isolate human DNA sequences, 100-200 bp in size, that are recognized by both partially purified and affinity purified proteins. Unlike other procedures designed to identify genomic target sequences, the method described does not require polymerase chain reaction or successive immunoprecipitations.     

Identification of a protein from escherichia coli whose synthesis appears to be triggered by the initiation of DNA replication

Using a DNA temperature sensitive initiation mutant to synchronize the replication and cell division cycle, we have compared proteins which are synthesized during a period of DNA arrest with those synthesized after return to permissive temperature. This work has led to the identification of a DNA-binding protein of 60–65,000 molecular weight (SDS-gel electrophoresis) whose synthesis appears to be triggered by the initiation event.     

Novel and Efficient Protocol for DNA Coating-Based Identification of DNA-Protein Interaction by Antibody-Mediated Immunodetection

Background:Studying protein-protein and protein-DNA interactions are prerequisites for the identification of function and mechanistic role of various proteins in the cell. Protocols for analyzing DNA-based Protein-Protein and Protein-DNA interactions are complicated and need to be simplified for efficient tracking of binding capabilities of various proteins to specific DNA molecules. Here, we demonstrated a simple yet efficient protocol for the identification of DNA coating-based Protein-DNA interaction using antibodymediated immunodetection.Methods:Briefly, we have coated specific DNA in the microtiter plate followed by incubating with protein lysate. Specific protein-DNA and/or protein-protein bind with DNA interactions are identified using specific fluorophore-conjugated antibodies. Antibodies are used to detect a protein that is bound to the DNA.Results:Fluorescent-based detection identifies the specific interaction between Protein-DNA with respect to coated DNA fragments. The protocol uses indirect conjugated antibodies and hence the technique is sensitive for effective identification of Protein-DNA interactions.Conclusion:Based on the results we conclude that the demonstrated protocol is simple, efficient and sensitive for identification of Protein-DNA interactions.Key Words: DNA coating, Lamin A, Protein-DNA interaction.