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

Abstract

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 .gtoreq. 100 S on alkaline sucrose gradients, which represents an estimated single strand size of .gtoreq. 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 EM 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 (.gtoreq. 80% of total labeled DNA) which is independent of the pulse period (1 min to 4 h). 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.