ABSTRACT
YB-1 is a DNA/RNA-binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA- and RNA-dependent events is determined by its localization in the cell. Distribution of YB-1 between the nucleus and the cytoplasm is known to be dependent on nuclear targeting and cytoplasmic retention signals located within the C-terminal portion of YB-1. Here, we report that YB-1 undergoes a specific proteolytic cleavage by the 20S proteasome, which splits off the C-terminal 105-amino-acid-long YB-1 fragment containing a cytoplasmic retention signal. Cleavage of YB-1 by the 20S proteasome in vitro appears to be ubiquitin- and ATP-independent, and is abolished by the association of YB-1 with messenger RNA. We also found that genotoxic stress triggers a proteasome-mediated cleavage of YB-1 in vivo and leads to accumulation of the truncated protein in nuclei of stressed cells. Endoproteolytic activity of the proteasome may therefore play an important role in regulating YB-1 functioning, especially under certain stress conditions.
Subject(s)
DNA Damage , Proteasome Endopeptidase Complex/metabolism , Y-Box-Binding Protein 1/metabolism , Adenosine Triphosphate/metabolism , Animals , Cell Nucleus/chemistry , Cell Nucleus/metabolism , DNA/drug effects , Drug Resistance, Neoplasm , Glycine/metabolism , Humans , Mice , Mutation , NIH 3T3 Cells , Rabbits , Tumor Cells, Cultured , Ubiquitin/metabolismABSTRACT
Ribosomal protein S1 has been identified in Thermus thermophilus ribosomes. The gene of ribosomal protein S1 from Thermus thermophilus has been cloned and overexpressed in Escherichia coli. A procedure for purification of the protein has been developed.
Subject(s)
Escherichia coli/genetics , Ribosomal Proteins/biosynthesis , Ribosomal Proteins/genetics , Thermus thermophilus/metabolism , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Escherichia coli/chemistry , Escherichia coli/metabolism , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Ribosomal Proteins/isolation & purification , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Thermus thermophilus/chemistryABSTRACT
Three novel DNA-binding proteins with apparent molecular masses of 7, 10 and 30 kDa have been isolated from the hyperthermophilic methanogen Methanopyrus kandleri. The proteins were identified using a blot overlay assay that was modified to emulate the high ionic strength intracellular environment of M.kandleri proteins. A 7 kDa protein, named 7kMk, was cloned and expressed in Escherichia coli. As indicated by CD spectroscopy and computer-assisted structure prediction methods, 7kMk is a substantially alpha-helical protein possibly containing a short N-terminal beta-strand. According to analytical gel filtration chromatography and chemical crosslinking, 7kMk exists as a stable dimer, susceptible to further oligomerization. Electron microscopy showed that 7kMk bends DNA and also leads to the formation of loop-like structures of approximately 43.5 +/- 3.5 nm (136 +/- 11 bp for B-form DNA) circumference. A topoisomerase relaxation assay demonstrated that looped DNA is negatively supercoiled under physiologically relevant conditions (high salt and temperature). A BLAST search did not yield 7kMk homologs at the amino acid sequence level, but based on a multiple alignment with ribbon-helix-helix (RHH) transcriptional regulators, fold features and self-association properties of 7kMk we hypothesize that it could be related to RHH proteins.
Subject(s)
DNA, Archaeal/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Euryarchaeota/genetics , Amino Acid Sequence , Chromatography, Gel , Circular Dichroism , Cloning, Molecular , Cross-Linking Reagents , DNA Topoisomerases, Type I/metabolism , DNA, Archaeal/chemistry , DNA, Archaeal/ultrastructure , DNA, Superhelical/chemistry , DNA, Superhelical/metabolism , DNA, Superhelical/ultrastructure , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/isolation & purification , Electrophoretic Mobility Shift Assay , Euryarchaeota/chemistry , Microscopy, Electron , Models, Biological , Molecular Sequence Data , Molecular Weight , Nucleic Acid Conformation , Nucleosomes/chemistry , Nucleosomes/genetics , Nucleosomes/metabolism , Osmolar Concentration , Protein Binding , Protein Structure, Quaternary , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Sequence Alignment , Sequence Analysis , SoftwareABSTRACT
Topoisomerase V (Topo V) is a type IB (eukaryotic-like) DNA topoisomerase. It was discovered in the hyperthermophilic prokaryote Methanopyrus kandleri and is the only topoisomerase with associated apurinic/apyrimidinic (AP) site-processing activities. The structure of Topo V in the free and DNA-bound states was probed by limited proteolysis at 37 degrees C and 80 degrees C. The Topo V protein is comprised of (i) a 44-kDa NH(2)-terminal core subdomain, which contains the active site tyrosine residue for topoisomerase activity, (ii) an immediately adjacent 16-kDa subdomain that contains degenerate helix-hairpin-helix (HhH) motifs, (iii) a protease-sensitive 18-kDa HhH "hinge" region, and (iv) a 34-kDa COOH-terminal HhH domain. Three truncated Topo V polypeptides comprising the NH(2)-terminal 44-kDa and 16-kDa domains (Topo61), the 44-, 16-, and 18-kDa domains (Topo78), and the COOH-terminal 34-kDa domain (Topo34) were cloned, purified, and characterized. Both Topo61 and Topo78 are active topoisomerases, but in contrast to Topo V these enzymes are inhibited by high salt concentrations. Topo34 has strong DNA-binding ability but shows no topoisomerase activity. Finally, we demonstrate that Topo78 and Topo34 possess AP lyase activities that are important in base excision DNA repair. Thus, Topo V has at least two active sites capable of processing AP DNA. The significance of multiple HhH motifs for the Topo V processivity is discussed.
