Abstract
Cytoplasmic intermediate filament (cIF) proteins interact strongly with single−stranded (ss) DNAs and RNAs, particularly with G−rich sequences. To test the hypothesis that this interaction depends on special nucleotide sequences and, possibly, higher order structures of ssDNA, a random mixture of mouse genomic ssDNA fragments generated by a novel "whole ssDNA genome PCR" technique via RNA intermediates was subjected to three rounds of affinity binding to in vitro reconstituted vimentin IFs at physiological ionic strength with intermediate PCR amplification of the bound ssDNA segments. Nucleotide sequence and computer folding analysis of the vimentin−selected fragments revealed an enrichment in microsatellites, predominantly of the (GT)n type, telomere DNA, and C/T−rich sequences, most of which, however, were incapable of folding into stable stem−loop structures. Because G−rich sequences were underrepresented in the vimentin−bound fraction, it had to be assumed that such sequences require intramolecular folding or lateral assembly into multistrand structures to be able to stably interact with vimentin, but that this requirement was inadequately fulfilled under the conditions of the selection experiment. For that reason, the few vimentin−selected G−rich ssDNA fragments and a number of telomere models were analyzed for their capacity to form inter− and intramolecular Gquadruplexes (G4 DNAs) under optimized conditions and to interact as such with vimentin and its type III relatives, glial fibrillary acidic protein, and desmin. Band shift assays indeed demonstrated differential binding of the cIF proteins to parallel four−stranded G4 DNAs and, with lower affinity, to bimolecular G'2 and unimolecular G'4 DNA configurations, whereby the transition regions from four− to single−strandedness played an additional role in the binding reaction. In this respect, the binding activity of cIF proteins was comparable with that toward other noncanonical DNA structures, like ds/ss DNA forks, triplex DNA, four−way junction DNA and Z−DNA, which also involve configurational transitions in their interaction with the filament proteins. Association of the cIF proteins with the corresponding nonfolded G−rich ssDNAs was negligible. Considering the almost universal involvement of ssDNA regions and G−quadruplexes in nuclear processes, including DNA transcription and recombination as well as telomere maintenance and dynamics, it is plausible to presume that cIF proteins as complementary constituents of the nuclear matrix participate in the cell− and tissue−specific regulation of these processes
