LONG-RANGE FRAGMENTATION OF THE EUKARYOTIC GENOME BY EXOGENOUS AND ENDOGENOUS NUCLEASES PROCEEDS IN A SPECIFIC FASHION VIA PREFERENTIAL DNACLEAVAGE AT MATRIX ATTACHMENT SITES

Small cell lung cancer cells (OC-NYH-VM) were permeabilized and treate d with different nucleases. The long-range distribution of DNA cleavag e sites in the amplified c-myc gene locus was then analyzed by pulsed field gel electrophoretic separation of the released 50-kilobase to 1- megabase DNA fragments followed by indirect end labeling. Exogenous DN ase I and nucleases specific for the single stranded DNA were found to generate similar nonrandom patterns of large DNA fragments. The cleav age sites were located close to or even colocalized with matrix attach ment regions, which were mapped independently using a recently develop ed procedure for DNA loop excision by DNA topoisomerase II-mediated DN A cleavage. Endogenous acidic nuclease with the properties of DNase II also digested DNA preferentially in proximity to the matrix attachmen t regions, generating characteristic patterns of excised DNA loops and their oligomers. A similar, although less specific, pattern of DNA fr agmentation was observed after incubation of permeabilized cells under conditions favoring the activity of endogenous neutral Ca2+- and Mg2-dependent nucleases, These findings are discussed in the context of t he current model of the spatial domain organization of eukaryotic geno me.