Email updates

Keep up to date with the latest news and content from BMC Cell Biology and BioMed Central.

Open Access Highly Accessed Research article

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

Zoltan Cseresnyes1, Ulf Schwarz2 and Catherine M Green1*

Author affiliations

1 Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ, UK

2 Leica Microsystems, CMS GmbH, Am Friedensplatz 3, 68165 Mannheim, Germany

For all author emails, please log on.

Citation and License

BMC Cell Biology 2009, 10:88  doi:10.1186/1471-2121-10-88

Published: 16 December 2009

Abstract

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.