Open AccessUse of site‐specific recombination as a probe of nucleoprotein complex formation in chromatin
Author(s) -
Schwikardi Micha,
Dröge Peter
Publication year - 2001
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.0014-2956.2001.02579.x
Subject(s) - chromatin , nucleoprotein , dna , histone , biology , nucleosome , chip sequencing , dna supercoil , chia pet , genetics , microbiology and biotechnology , chip on chip , site specific recombination , hmg box , linker dna , computational biology , recombination , dna binding protein , recombinase , dna replication , gene , transcription factor
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.