title: Type III intermediate filament proteins interact with four-way junction DNA and facilitate its cleavage by the junction-resolving enzyme T7 endonuclease I.
abstract: The isolation from proliferating mouse and human embryo fibroblasts of SDS-stable crosslinkage products of vimentin with DNA fragments containing inverted repeats capable of cruciform formation under superhelical stress and the competitive effect of a synthetic Holliday junction on the binding of cytoplasmic intermediate filament (cIF) proteins to supercoiled DNA prompted a detailed investigation of the proteins' capacity to associate with four-way junction DNA and to influence its processing by junction-resolving endonucleases. Electrophoretic mobility shift analysis of reaction products obtained from vimentin and Holliday junctions under varying ionic conditions revealed efficient complex formation of the filament protein not only with the unstacked, square-planar configuration of the junctions but also with their coaxially stacked X-conformation. Glial fibrillary acidic protein (GFAP) was less efficient and desmin virtually inactive in complex formation. Electron microscopy showed binding of vimentin tetramers or octamers almost exclusively to the branchpoint of the Holliday junctions under physiological ionic conditions. Even at several hundredfold molar excess, sequence-related single- and double-stranded DNAs were unable to chase Holliday junctions from their complexes with vimentin. Vimentin also stimulated bacteriophage T7 endonuclease I in introducing single-strand cuts diametrically across the branchpoint and thus in the resolution of the Holliday junctions. This effect is very likely due to vimentin-induced structural distortion of the branchpoint, as suggested by the results of hydroxyl radical footprinting of Holliday junctions in the absence and the presence of vimentin. Moreover, vimentin, and to a lesser extent GFAP and desmin, interacted with the cruciform structures of inverted repeats inserted into a supercoiled vector plasmid, thereby changing their configuration via branch migration and sensibilizing them to processing by T7 endonuclease I. This refers to both plasmid relaxation caused by unilateral scission and, particularly, linearization via bilateral scission at primary and cIF protein-induced secondary cruciform branchpoints that were identified by T7 endonuclease I footprinting. cIF proteins share these activities with a variety of other architectural proteins interacting with and structurally modulating four-way DNA junctions. In view of the known and hypothetical functions of four-way DNA junctions and associated protein factors in DNA metabolism, cIF proteins as complementary nuclear matrix proteins may play important roles in such nuclear matrix-associated processes as DNA replication, recombination, repair, and transcription, with special emphasis on both the preservation and evolution of the genome.
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: Michael
authors.initials: M
authors.lastname: Sabasch
authors.forename: Peter
authors.initials: P
authors.lastname: Traub
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000378:metabolism; D003893:Desmin / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014746:Vimentin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D003893:Desmin; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D046911:Macromolecular Substances; D014746:Vimentin; D004247:DNA; D003850:Deoxyribonuclease I