Pancreatic DNAase cleavage sites in nuclei

Abstract

The DNA of nuclei is cleaved by a variety of nucleases in such a way that the cuts on a given strand are always separated by an integral multiple of 10 nucleotides. However, the spacing between cutting sites on opposite strands is not known for any nuclease. In this paper, we describe the determination of the spacing, or stagger, between cuts on opposite strands produced by the action of pancreatic DNAase (DNAase I) on nuclei. When nuclei are digested with DNAase I and the resultant DNA is analyzed by gel electrophoresis without prior denaturation, a complex pattern of bands is observed. A method which gives better than 90% recovery of DNA from polyacrylamide gels was used to isolate the individual fractions corresponding to these bands. The structure of the fractions was then determined using single-strand-specific nuclease to digest single-stranded "tails" and using DNA polymerases to extend recessed 3'-OH termini of partially duplex regions. Our results show that each component consists of a double-stranded region terminating in single-stranded tails at both ends. Although both chains of every duplex are 10-n nucleotides long (n integer), the chains are never completely paired. The experiments with DNA polymerase show an abundance of structures in which the 3'-OH termini of these duplexes are recessed by 8 nucleotides, and by inference, there must be structures with 5'-P termini recessed by 2 or 12 nucleotides. Thus DNAase I acts on nuclei to produce DNA with staggered cuts on opposite strands, separated by (10-n + 8) and (10-n + 2) base pairs (with 5'-P and 3'-OH termini extending, respectively). Two classes of models of DNA folding in the nucleosome have been proposed by other investigators to account for the presence of DNAase I cleavage sites at 10-n intervals along each DNA chain. One class of models leads to the prediction that cuts should either be unstaggered or separated by 10 nucleotides, while the other class is consistent with staggers of 6 and 4 nucleotides. Neither prediction is verified by our data; however, all these models may be made consistent with the results by assuming that the enzyme's site of recognition on nucleosomal DNA is not the same as its site of cleavage.