Localization and dynamics of small circular DNA in live mammalian nuclei

• Published in: Nucleic acids research Vol. 32; no. 8; pp. 2642 - 2651
• Main Authors: Mearini, Giulia, Nielsen, Peter E., Fackelmayer, Frank O.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 2004; Oxford Publishing Limited (England)
• Subjects: Animals; Biological Transport; Cell Nucleus - genetics; Cell Nucleus - metabolism; Cell Nucleus Structures - genetics; Cercopithecus aethiops; COS Cells; DNA, Circular - administration & dosage; DNA, Circular - analysis; DNA, Circular - chemistry; DNA, Circular - metabolism; Kinetics; Microinjections; Nuclear Matrix - chemistry; Plasmids - administration & dosage; Plasmids - analysis; Plasmids - metabolism
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkh587

While genomic DNA, packaged into chromatin, is known to be locally constrained but highly dynamic in the nuclei of living cells, little is known about the localization and dynamics of small circular DNA molecules that invade cells by virus infection, application of gene therapy vectors or experimental transfection. To address this point, we have created traceable model substrates by direct labeling of plasmid DNA with fluorescent peptide nucleic acids, and have investigated their fate after microinjection into living cells. Here, we report that foreign DNA rapidly undergoes interactions with intranuclear structural sites that strongly reduce its mobility and restrict the DNA to regions excluding nucleoli and nuclear bodies such as PML bodies. The labeled plasmids partially co‐localize with SAF‐A, a well characterized marker protein for the nuclear ‘scaffold’ or ‘matrix’, and are resistant towards extraction by detergent and, in part, elevated salt concentrations. We show that the localization and the low mobility of plasmids is independent of the plasmid sequence, and does not require the presence of either a scaffold attachment region (SAR) DNA element or a functional promoter.