Use of site-specific recombination as a probe of nucleoprotein complex formation in chromatin

DNA transactions in eukaryotes require that proteins gain access to target sequences packaged in chromatin. Further, interactions between distinct nuc leoprotein 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 s tructures of different complexities at more than 30 genomic loci. The dynam ic nature of chromatin permitted protein-DNA and DNA-DNA interactions for s ites of at least 34 by in length. However, the assembly of higher-order nuc leoprotein structures on targets spanning 114 by was impaired. This impedim ent was maintained over at least 72 h and was not affected by the transcrip tional status of chromatin nor by inhibitors of histone deacetylases and to poisomerases. Our findings suggest that nucleosomal linker-sized DNA segmen ts become accessible within hours for protein binding due to the dynamic na ture 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 Fl p are proficient in eukaryotic chromatin.