Long-range fragmentation of the eukaryotic genome by exogenous and endogenous nucleases proceeds in a specific fashion via preferential DNA cleavage at matrix attachment sites.
Small cell lung cancer cells (OC-NYH-VM) were permeabilized and treated with different nucleases. The long-range distribution of DNA cleavage 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 DNase I and nucleases specific for the single-stranded DNA were found to generate similar nonrandom patterns of large DNA fragments. The cleavage sites were located close to or even colocalized with matrix attachment regions, which were mapped independently using a recently developed procedure for DNA loop excision by DNA topoisomerase II-mediated DNA cleavage. Endogenous acidic nuclease with the properties of DNase II also digested DNA preferentially in proximity to the matrix attachment regions, generating characteristic patterns of excised DNA loops and their oligomers. A similar, although less specific, pattern of DNA fragmentation was observed after incubation of permeabilized cells under conditions favoring the activity of endogenous neutral Ca(2+)- and Mg(2+)-dependent nucleases. These findings are discussed in the context of the current model of the spatial domain organization of eukaryotic genome.
Gromova
I I
II
Department of Molecular Biology, University of Aarhus, Denmark.
Nielsen
O F
OF
Razin
S V
SV
D002118:Calcium / Q000494:pharmacology; D018288:Carcinoma, Small Cell; D002463:Cell Membrane Permeability; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D003850:Deoxyribonuclease I / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism*; D016259:Genes, myc; D016678:Genome; D006801:Humans; D008175:Lung Neoplasms; D008274:Magnesium / Q000494:pharmacology; D008836:Micrococcal Nuclease / Q000378:metabolism; D015530:Nuclear Matrix; D010316:Particle Size; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D004247:DNA; D004720:Endonucleases; D003850:Deoxyribonuclease I; D015719:Single-Strand Specific DNA and RNA Endonucleases; D008836:Micrococcal Nuclease; D008274:Magnesium; D002118:Calcium