Visualization and Interpretation of Eukaryotic DNA Replication Intermediates In Vivo by Electron Microscopy

Abstract

The detailed understanding of the DNA replication process requires structural insight. The combination of psoralen cross-linking and electron microscopy has been extensively exploited to reveal the fine architecture of in vivo DNA replication intermediates. This approach proved instrumental to uncover the basic mechanisms of DNA duplication, as well as the perturbation of this process by various forms of replication stress. The replication structures are stabilized in vivo (by psoralen cross-linking) prior to extraction and enrichment procedures, allowing their visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize and interpret in vivo replication intermediates of genomic DNA, extracted from budding yeast, Xenopus egg extracts, or cultured mammalian cells.