Analysis of replication factories in human cells by super-resolution light microscopy.
BACKGROUND
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. 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.


RESULTS
Using 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.


CONCLUSIONS
These 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.
Cseresnyes
Zoltan
Z
Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.
Schwarz
Ulf
U
Green
Catherine M
CM
D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication; D005347:Fibroblasts / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008853:Microscopy; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism
D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A