ABSTRACT
Ten short DNA fragments have been selected from a library of the nuclear matrix-attached DNA (nmDNA) from chicken erythrocytes by their ability to hybridize with the fraction of chicken replication origins isolated by nascent DNA strand extrusion. The primary structure of these fragments has been determined. Five of the sequences contained a topoisomerase II recognition site. Most of the studied DNA fragments also have a common eight-nucleotide motif, GCAGACCG/A. A sequence-specific DNA-binding protein with a MW of 55 kDa that interacted with this motif has been identified. Some of the cloned DNA fragments promoted an increased level of transient plasmid replication in transfected chicken cells. The ability of plasmid bearing nmDNA fragments to replicate correlated directly with their ability to target plasmids to the nuclear matrix compartment.
Subject(s)
DNA Replication , DNA/metabolism , Nuclear Matrix/metabolism , Animals , Base Sequence , Binding Sites , Cells, Cultured , Chickens , Cloning, Molecular , DNA/chemistry , DNA/genetics , Erythrocytes/metabolism , Molecular Sequence Data , PlasmidsABSTRACT
Most cells of higher eukaryotes may maintain several rounds of replication of circular DNA. Efficiency of replication is usually low, and depends on the length of the circular DNA rather than on the sequence context. We have isolated and characterized several short DNA fragments that form structural sites of attachment to the nuclear matrix (nmDNA) in chicken cells, and tested whether they would enhance autonomous replication of DNA in chicken cells as compared to the vector DNA. Indeed, a several-fold increase in a short-term replication efficiency was detected using a semi-conservative replication and a DpnI-resistance assay. Most of the cloned matrix-associated fragments were recovered in the nuclear matrix fraction when introduced into cultured chicken cells as a circular DNA. The data obtained suggest that the observed enhancement in the replication efficiency of the circular DNA may be due to their recruitment to the nuclear matrix by the nmDNA.
Subject(s)
DNA Replication/genetics , DNA/genetics , Erythroblasts/metabolism , Nuclear Matrix/metabolism , Animals , Base Sequence , Cell Line , Chickens , Cloning, Molecular , DNA/metabolism , Erythroblasts/cytology , Molecular Sequence Data , Plasmids/geneticsABSTRACT
The sequence-specific DNA-binding protein factor F6, which binds upstream of the cluster of the chicken alpha-globin genes, has previously been found to interact with a DNA fragment containing a replication origin and a nuclear matrix binding site. This protein has been partially characterized. Based on its molecular weight and binding affinity, F6 belongs to a family of GATA proteins, the chicken equivalent of transcription factor NFE-1. An oligonucleotide including the binding site for F6 competes for binding of the above-mentioned DNA fragment to the nuclear matrix. This indicates an involvement of this protein in the interaction between DNA and the nuclear matrix.
