Supercoiled loops and eucaryotic DNA replication

Abstract

Recent studies have indicated that the DNA of the eucaryotic nucleus is organized in the form of supercoiled loops. We show here that after depleting interphase nuclei of histones and other nuclear proteins by treatment with nonionic detergent and high salt, intact (unnicked) loops of DNA can be visualized as a halo surrounding a nuclear skeleton or matrix. This halo of DNA loops can be reversibly wound using various concentrations of ethidium bromide and is irreversibly unwound when the DNA is gently nicked with DNAase I or exposure to ultraviolet light. These structures, comprising a halo of unwound DNA loops anchored to a nuclear matrix, provided us with a way in which to examine the relationship of DNA replication to the DNA loops. 3T3 cells were pulse-labeled with 3H-thymidine for various periods and autoradiography was performed on the matrix-halo structures. It was found that after a 1 min pulse, >80% of the autoradiographic grains representing newly replicated DNA were found within the nuclear matrix even though the surrounding halo region contained 80% of the total nuclear DNA. With longer pulse times, the autoradiographic grains could be seen to move progressively outward from the nuclear matrix into the halo region. Over 70% of the autoradiographic grains could be chased out of the central matrix into the surrounding halo if the 1 min pulse was followed by a 1 hr incubation with excess unlabeled thymidine. These findings indicate that DNA replication occurs at fixed sites at the base of the loops and suggests that the replicating DNA loops are motile with respect to their nuclear matrix anchorage sites.