Mapping DNAase l-susceptible sites in nucleosomes labeled at the 5' ends

Abstract

We have used lambda-32P-ATP and polynucleotide kinase to label stoichiometrically the 5' ends of DNA in intact isolated HeLa cell nucleosomes. DNA is not nicked or degraded during the modification reaction. We have used these modified nucleosomes to study both the distribution and the relative availability of sites within the nucleosome which are susceptible to digestion by DNAase l. The results show that the nucleosome contains a potential cleavage site every 10 nucleotides, with the exception of the site 80 nucleotides from the 5' end. Favored cleavage sites are located 20, 40, 50, 100, 120, and 130 nucleotides from the 5' end; sites 30 and 110 nucleotides from the 5' end are strongly disfavored, while the potential site 80 nucleotides from the 5' end is virtually never cleaved. These findings provide constraints for models of histone-DNA interactions within the chromatin subunit.