ABSTRACT
The tightly bound proteins (TBPs), a protein group that remains attached to DNA either covalently or noncovalently after deproteinization, have been found in numerous eukaryotic species. Some TBPs isolated from mammalian and yeast cells possess phosphatase or kinase activity. The aim of this study was to characterize further TBPs in barley (Hordeum vulgare) cells. The spectra of TBPs varied in different organs of barley shoots (first leaves, coleoptile, and roots) and at different developmental stages of the plant. Some barley TBPs manifested phosphatase, probably Ser/Thr or dual Ser/Thr/Tyr activity. MALDI-TOF mass spectrometry of barley TBPs identified several proteins involved in chromatin rearrangement and regulation processes, including transcription factors, serpins, protein phosphatases and protein kinases, RNA helicases, and DNA topoisomerase II.
Subject(s)
DNA, Plant/metabolism , DNA-Binding Proteins/metabolism , Hordeum/growth & development , Phosphoric Monoester Hydrolases/metabolism , Plant Proteins/metabolism , Chromatin Assembly and Disassembly/physiology , Cotyledon/enzymology , Cotyledon/genetics , Cotyledon/growth & development , DNA Topoisomerases, Type II/analysis , Hordeum/enzymology , Hordeum/genetics , Plant Leaves/enzymology , Plant Leaves/genetics , Plant Leaves/growth & development , Plant Proteins/analysis , Plant Roots/enzymology , Plant Roots/genetics , Plant Roots/growth & development , Protein Kinases/analysis , RNA Helicases/analysis , Serpins/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Tightly bound to DNA proteins (TBPs) are a protein group that remains attached to DNA after its deproteinization by phenol, chloroform or salting-out. TBP are bound to DNA with covalent phosphotriester or non-covalent ion and hydrogen bonds. They appear to be a vast protein group involved in numerous intranuclear processes. The TBPs fraction co-purified with DNA deproteinized by mild procedures is extremely heterogeneous, tissue and species-specific. The protein fraction co-purified with DNA after harsh deproteinization procedures appears to be formed from few polypeptides common to different species and tissues. Interaction sites between DNA and TBPs depend on the physiological status of the cell. The binding sites of TBPs to DNA do not co-localize with the nuclear matrix attachment regions. We hypothesize that TBPs form a universal substrate for intranuclear processes.
Subject(s)
Cell Nucleus/metabolism , DNA-Binding Proteins/metabolism , Animals , DNA-Binding Proteins/chemistry , Models, Biological , Organ Specificity , Phosphoric Monoester Hydrolases/metabolism , Serpins/metabolism , Species Specificity , Transcription, GeneticABSTRACT
Proteins tightly bound to DNA (TBP) comprise a group of proteins that remain bound to DNA after usual deproteinization procedures such as salting out and treatment with phenol or chloroform. TBP bind to DNA by covalent phosphotriester and noncovalent ionic and hydrogen bonds. Some TBP are conservative, and they are usually covalently bound to DNA. However, the TBP composition is very diverse and significantly different in different tissues and in different organisms. TBP include transcription factors, enzymes of the ubiquitin-proteasome system, phosphatases, protein kinases, serpins, and proteins of retrotransposons. Their distribution within the genome is nonrandom. However, the DNA primary structure or DNA curvatures do not define the affinity of TBP to DNA. But there are repetitive DNA sequences with which TBP interact more often. The TBP distribution within genes and chromosomes depends on a cell's physiological state, differentiation type, and stage of organism development. TBP do not interact with DNA in the sites of its association with nuclear matrix and most likely they are not components of the latter.
