Abstract
BackgroundDNA replication in human cells is performed in discrete sub-nuclear locations known as replication foci or factories. These factories form in the nucleus during S phase and are sites of DNA synthesis and high local concentrations of enzymes required for chromatin replication. Why these structures are required, and how they are organised internally has yet to be identified. It has been difficult to analyse the structure of these factories as they are small in size and thus below the resolution limit of the standard confocal microscope. We have used stimulated emission depletion (STED) microscopy, which improves on the resolving power of the confocal microscope, to probe the structure of these factories at sub-diffraction limit resolution.ResultsUsing immunofluorescent imaging of PCNA (proliferating cell nuclear antigen) and RPA (replication protein A) we show that factories are smaller in size (approximately 150 nm diameter), and greater in number (up to 1400 in an early S- phase nucleus), than is determined by confocal imaging. The replication inhibitor hydroxyurea caused an approximately 40% reduction in number and a 30% increase in diameter of replication factories, changes that were not clearly identified by standard confocal imaging.ConclusionsThese measurements for replication factory size now approach the dimensions suggested by electron microscopy. This agreement between these two methods, that use very different sample preparation and imaging conditions, suggests that we have arrived at a true measurement for the size of these structures. The number of individual factories present in a single nucleus that we measure using this system is greater than has been previously reported. This analysis therefore suggests that each replication factory contains fewer active replication forks than previously envisaged.
Highlights
DNA replication in human cells is performed in discrete sub-nuclear locations known as replication foci or factories
As previously demonstrated [9,25,16], PCNA and RPA were localised in focal patterns in nuclei that were labelled with EdU and actively replicating DNA
In these cells the PCNA and RPA patterns closely follow that of EdU, and the colocalisation is striking but not complete. This is expected as PCNA is loaded at primer-template junctions, and RPA binds template DNA, whereas the EdU is incorporated as DNA is synthesised
Summary
DNA replication in human cells is performed in discrete sub-nuclear locations known as replication foci or factories These factories form in the nucleus during S phase and are sites of DNA synthesis and high local concentrations of enzymes required for chromatin replication. In mammalian cells chromosomal replication occurs at discrete nuclear sites, known as replication foci or replication factories [6,7] These structures form transiently in the nucleus and are the sites of DNA synthesis and high local concentrations of replication proteins [8,9,10,11,12,13,14,15]. The internal organisation of these replication factories and the possible protein-protein interactions dictating and controlling their formation, persistence and disassembly remain unclear
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