title: Interaction in vitro of type III intermediate filament proteins with Z-DNA and B-Z-DNA junctions.
abstract: The selection of DNA fragments containing simple d(GT)(n) and composite d(GT)(m). d(GA)(n) microsatellites during affinity binding of mouse genomic DNA to type III cytoplasmic intermediate filaments (cIFs) in vitro, and the detection of such repeats, often as parts of nuclear matrix attachment region (MAR)-like DNA, in SDS-stable DNA-vimentin crosslinkage products isolated from intact fibroblasts, prompted a detailed study of the interaction of type III cIF proteins with left-handed Z-DNA formed from d(GT)(17) and d(CG)(17) repeats under the topological tension of negatively supercoiled plasmids. Although d(GT)(n) tracts possess a distinctly lower Z-DNA-forming potential than d(CG)(n) tracts, the filament proteins produced a stronger electrophoretic mobility shift with a plasmid carrying a d(GT)(17) insert than with plasmids containing different d(CG)(n) inserts, consistent with the facts that the B-Z transition of d(GT)(n) repeats requires a higher negative superhelical density than that of d(CG)(n) repeats and the affinity of cIF proteins for plasmid DNA increases with its superhelical tension. That both types of dinucleotide repeat had indeed undergone B-Z transition was confirmed by S1 nuclease and chemical footprinting analysis of the plasmids, which also demonstrated efficient protection by cIF proteins from nucleolytic and chemical attack of the Z-DNA helices as such, as well as of the flanking B-Z junctions. The analysis also revealed sensibilization of nucleotides in the center of one of the two strands of a perfect d(CG)(17) insert toward S1 nuclease, indicating cIF protein-induced bending of the repeat. In all these assays, vimentin and glial fibrillary acidic protein (GFAP) showed comparable activities, versus desmin, which was almost inactive. In addition, vimentin and GFAP exhibited much higher affinities for the Z-DNA conformation of brominated, linear d(CG)(25) repeats than for the B-DNA configuration of the unmodified oligonucleotides. While double-stranded DNA was incapable of chasing the Z-DNA from its protein complexes, and Holliday junction and single-stranded (ss)DNA were distinguished by reasonable competitiveness, phosphatidylinositol (PI) and, particularly, phosphatidylinositol 4,5-diphosphate (PIP(2)) turned out to be extremely potent competitors. Because PIP(2) is an important member of the nuclear PI signal transduction cascade, it might exert a regulatory influence on the binding of cIF proteins to Z- and other DNA conformations. From this interaction of cIF proteins with Z- and bent DNA and their previously detected affinities for MAR-like, ss, triple helical, and four-way junction DNA, it may be concluded that the filament proteins play a general role in such nuclear matrix-associated processes as DNA replication, recombination, repair, and transcription.
authors.affiliation: Max-Planck Institut für Zellbiologie, Rosenhof, 68526 Ladenburg, Germany.
authors.forename: Guohong
authors.initials: G
authors.lastname: Li
authors.forename: Genrich V
authors.initials: GV
authors.lastname: Tolstonog
authors.forename: Peter
authors.initials: P
authors.lastname: Traub
mesh_terms: D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D001966:Bromine / Q000737:chemistry; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018983:DNA Footprinting / Q000379:methods; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D003893:Desmin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018900:Dinucleotide Repeats; D024202:Electrophoretic Mobility Shift Assay; D005904:Glial Fibrillary Acidic Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015530:Nuclear Matrix / Q000235:genetics / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014746:Vimentin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D003893:Desmin; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D014746:Vimentin; D004247:DNA; D001966:Bromine