No abstract

Biochemical fractionation was combined with high resolution electron microscopic autoradiography to study the localization in rat liver nuclear matrix of attached DNA fragments, in vivo replicated DNA, and in vitro synthesized DNA. In particular, we determined the distribution of these DNA components with the peripheral nuclear lamina versus more internally localized structural elements of isolated nuclear matrix. Autoradiography demonstrated that the bulk of in vivo newly replicated DNA associated with the nuclear matrix (71%) was found within internal matrix regions. A similar interior localization was observed in isolated nuclei and in situ in whole liver tissue. Likewise, isolated nulcear lamina contained only a small amount (12%) of the total matrix-bound, newly replicated DNA. The structural localization of matrix-bound DNA fragments was examined following long-term in vivo labeling of the DNA. The radioactive DNA fragments were found predominantly within interior regions of the matrix structure (77%), and isolated nuclear lamina contained

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.