title: Use of site-specific recombination as a probe of nucleoprotein complex formation in chromatin.
abstract: DNA transactions in eukaryotes require that proteins gain access to target sequences packaged in chromatin. Further, interactions between distinct nucleoprotein complexes are often required to generate higher-order structures. Here, we employed two prokaryotic site-specific recombination systems to investigate how chromatin packaging affects the assembly of nucleoprotein structures of different complexities at more than 30 genomic loci. The dynamic nature of chromatin permitted protein-DNA and DNA-DNA interactions for sites of at least 34 bp in length. However, the assembly of higher-order nucleoprotein structures on targets spanning 114 bp was impaired. This impediment was maintained over at least 72 h and was not affected by the transcriptional status of chromatin nor by inhibitors of histone deacetylases and topoisomerases. Our findings suggest that nucleosomal linker-sized DNA segments become accessible within hours for protein binding due to the dynamic nature of chromatin. Longer segments, however, appear refractory for complete occupancy by sequence-specific DNA-binding proteins. The results thus also provide an explanation why simple recombination systems such as Cre and Flp are proficient in eukaryotic chromatin.
authors.affiliation: Institute of Genetics, University of Cologne, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Schwikardi
authors.forename: P
authors.initials: P
authors.lastname: Dröge
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002843:Chromatin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006224:Cricetinae; D056572:Histone Deacetylase Inhibitors; D019426:Integrases; D046911:Macromolecular Substances; D009698:Nucleoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D045522:Recombinases; D011995:Recombination, Genetic; D059003:Topoisomerase Inhibitors; D019895:Transposases; D014764:Viral Proteins
chemical_list: D002843:Chromatin; D056572:Histone Deacetylase Inhibitors; D046911:Macromolecular Substances; D009698:Nucleoproteins; D045522:Recombinases; D059003:Topoisomerase Inhibitors; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases; D019895:Transposases