Isolation and characterization of rat liver nuclear matrices containing high molecular weight deoxyribonucleic acid

Rat liver nuclear matrices isolated by a method which limits DNA degradation contain a major portion of the total nuclear DNA. A majority of the DNA sediments at greater than or equal 100 S on alkaline sucrose gradients, which represents an estimated single strand size of greater than or equal to 500 kilobases. These DNA-rich matrices were virtually identical with previously isolated DNA-depleted matrices in recovery of total nuclear protein and overall polypeptide composition on sodium dodecyl sulfate-acrylamide gels. Thin-sectioning electron microscopy revealed a structure similar to the DNA-depleted matrices with the addition of a prominent meshwork of DNA fibrils extended throughout the matrix interior. In vivo labeling of regenerating livers showed a continuous association of newly replicated DNA with DNA-rich matrices (greater than or equal to 80% of total labeled DNA) which is independent of the pulse period (1 min to 4 h). Moreover, the matrix-associated DNA is highly enriched in replicating intermediates after a 1-min in vivo pulse including a small amount of the primary Okazaki fragments. The matrix-associated replicating intermediates (4-50 S) are effectively chased into DNA of replicon size and larger (100 S) following a 1-h pulse. DNA-rich nuclear matrices may therefore provide a useful in vitro system for studying DNA replication in correlation with the higher order, intranuclear arrangement of eukaryotic DNA.