ABSTRACT
The Ku antigen consists of two subunits of 70 and 83 kDa and is endowed with both duplex DNA end-binding capacity and helicase activity (human DNA helicase II). HeLa Ku can be isolated from in vitro cultured human cells uniquely as a heterodimer, and the subunits can be separated by electrophoresis only under denaturing conditions. To dissect the molecular functions of the two subunits of the heterodimer, we have cloned and expressed their cDNAs separately in Escherichia coli. The two activities of Ku (DNA binding and unwinding) were reconstituted by mixing and refolding both subunits in equimolar amounts (Tuteja, N., Tuteja, R., Ochem, A., Taneja, P., Huang, N-W., Simoncsits, A., Susic, S., Rahman, K., Marusic, L., Chen, J., Zang, J., Wang, S., Pongor, S., and Falaschi, A. (1994) EMBO J. 13, 4991-5001). Renaturation of the separate subunits can be achieved in the presence of a synthetic solubilizing and stabilizing agent, dimethyl ethylammonium propane sulfonate (NDSB 195). The helicase activity of the Ku protein resides uniquely in the 70-kDa subunit, whereas the DNA end-binding activity can be reconstituted only through renaturation of the two subunits in the heterodimeric form and is practically absent in the separate subunits. The 83-kDa subunit, when refolded in the absence of the 70-kDa subunit, forms homodimers unable to unwind DNA and bind duplex ends. The three separate species (heterodimer, 70-kDa subunit, and 83-kDa subunit homodimer) all have ssDNA-dependent ATPase activity.
Subject(s)
Adenosine Triphosphatases/metabolism , Antigens, Nuclear , Autoantigens/metabolism , DNA Helicases , DNA Repair , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Adenosine Triphosphate/metabolism , HeLa Cells , Humans , Kinetics , Ku Autoantigen , Protein Conformation , Protein Denaturation , Protein Folding , Recombinant Proteins/metabolism , SolubilityABSTRACT
A novel ATP-dependent DNA unwinding enzyme, called human DNA helicase VI (HDH VI), was purified to apparent homogeneity from HeLa cells and characterized. From 327 g of cultured cells, 0.44 mg of pure enzyme was recovered, free of DNA polymerase, ligase, topoisomerase, nicking and nuclease activities. The enzyme behaves as a monomer having an M(r) of 128 kDa, whether determined with SDS-PAGE, or in native conditions. Photoaffinity labelling with [alpha-32P]ATP labelled the 128 kDa protein. Only ATP or dATP hydrolysis supports the unwinding activity for which a divalent cation (Mg2+ > Mn2+) is required. HDH VI unwinds exclusively DNA duplexes with an annealed portion < 32 bp and prefers a replication fork-like structure of the substrate. It cannot unwind blunt-end duplexes and is inactive also on DNA-RNA or RNA-RNA hybrids. HDH VI unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand.
Subject(s)
DNA Helicases/isolation & purification , DNA/metabolism , Adenosine Triphosphate/metabolism , Affinity Labels , Cations, Divalent , Chemical Phenomena , Chemistry, Physical , DNA Helicases/chemistry , DNA Helicases/metabolism , DNA, Single-Stranded , Deoxyadenine Nucleotides/metabolism , Enzyme Stability , HeLa Cells , Hot Temperature , Humans , Hydrogen-Ion Concentration , Hydrolysis , Magnesium/pharmacology , Manganese/pharmacology , Photochemistry , RNA/metabolism , Substrate SpecificityABSTRACT
The cDNA encoding human DNA helicase IV (HDH IV), a 100-kDa protein which unwinds DNA in the 5' to 3' direction with respect to the bound strand, was cloned and sequenced. It was found to be identical to the human cDNA encoding nucleolin, a ubiquitous eukaryotic protein essential for pre-ribosome assembly. HDH IV/nucleolin can unwind RNA-RNA duplexes, as well as DNA-DNA and DNA-RNA duplexes. Phosphorylation of HDH IV/nucleolin by cdc2 kinase and casein kinase II enhanced its unwinding activity in an additive way. The Gly-rich C-terminal domain possesses a limited ATP-dependent duplex-unwinding activity which contributes to the helicase activity of HDH IV/nucleolin.
Subject(s)
Adenosine Triphosphatases/genetics , DNA Helicases/genetics , DNA-Binding Proteins , Escherichia coli Proteins , Nuclear Proteins/genetics , Phosphoproteins/genetics , RNA-Binding Proteins , Adenosine Triphosphatases/immunology , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Blotting, Western , CDC2 Protein Kinase/metabolism , Casein Kinase II , Cloning, Molecular , DNA/metabolism , DNA Helicases/immunology , DNA Helicases/metabolism , DNA, Complementary/genetics , HeLa Cells/enzymology , Humans , Nuclear Proteins/immunology , Nuclear Proteins/metabolism , Phosphoproteins/immunology , Phosphoproteins/metabolism , Phosphorylation , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/metabolism , Sequence Homology , NucleolinABSTRACT
Human DNA helicase III, a novel DNA unwinding enzyme, has been purified to apparent homogeneity from nuclear extracts of HeLa cells and characterized. The activity was measured by using a strand displacement assay with a 32P labeled oligonucleotide annealed to M13 ssDNA. From 305 grams of cultured cells 0.26 mg of pure protein was isolated which was free of DNA topoisomerase, ligase, nicking and nuclease activities. The apparent molecular weight is 46 kDa on SDS polyacrylamide gel electrophoresis. The enzyme shows also DNA dependent ATPase activity and moves unidirectionally along the bound strand in 3' to 5' direction. It prefers ATP to dATP as a cofactor and requires a divalent cation (Mg2+ > Mn2+). Helicase III cannot unwind either blunt-ended duplex DNA or DNA-RNA hybrids and requires more than 84 bases of ssDNA in order to exert its unwinding activity. This enzyme is unique among human helicases as it requires a fork-like structure on the substrate for maximum activity, contrary to the previously described human DNA helicases I and IV, (Tuteja et al. Nucleic Acids Res. 18, 6785-6792, 1990; Tuteja et al. Nucleic Acids Res. 19, 3613-3618, 1991).
