Nucleoskeleton and initiation of DNA replication in metazoan cells

To determine whether or not initiation sites for DNA replication in mammali an cells are defined by association with nuclear structure, attachments bet ween the nucleoskeleton and the hamster DHFR gene initiation zone were exam ined. Nucleoskeletons were prepared by encapsulating cells in agarose and t hen extracting them with a nonionic detergent in a physiological buffer. Th e fraction of DNA that remained following endonuclease digestion was resist ant to salt, sensitive to Sarkosyl, and essentially unchanged by glutaralde hyde crosslinking, Although newly replicated DNA was preferentially attache d to the nucleoskeleton, no specific sequence was preferentially attached w ithin a 65 kb locus containing the DHFR gene, two origins of bi-directional replication and at least one nuclear matrix attachment region. Instead, th e entire region went from preferentially unattached to preferentially attac hed as cells progressed from G(1) to late S-phase, Thus, initiation sites i n mammalian chromosomes are not defined by attachments to the nucleoskeleto n. To further assess the relationship between the nucleoskeleton and DNA re plication, plasmid DNA containing the DHFR initiation region was replicated in a Xenopus egg extract. All of the DNA associated with the nucleoskeleto n prior to S-phase without preference for a particular sequence and was rel eased upon mitosis, However, about half of this DNA was trapped rather than bound to the nucleoskeleton, Thus, attachments to the nncleoskeleton can f orm in the absence of either DNA replication or transcription, but if they are required for replication, they are not maintained once replication is c ompleted.