Electron microscopy methods for studying in vivo DNA replication intermediates.
The detailed understanding of the DNA replication process requires structural insight. The combination of psoralen crosslinking 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 genotoxic treatments. The replication structures need to the stabilized in vivo (by psoralen crosslinking) prior to extraction and enrichment procedures, finally leading to the visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize in vivo replication intermediates of genomic DNA, extracted from budding yeast or cultured mammalian cells.
Lopes
Massimo
M
Institute of Molecular Cancer Research, University of Zürich, Zürich, Switzerland.
D000818:Animals; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D003432:Cross-Linking Reagents; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry / Q000648:ultrastructure*; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D011564:Furocoumarins; D016681:Genome, Fungal; D006801:Humans; D046529:Microscopy, Electron, Transmission / Q000295:instrumentation / Q000379:methods*; D009691:Nucleic Acid Denaturation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D014307:Trioxsalen
D003432:Cross-Linking Reagents; D004271:DNA, Fungal; D011564:Furocoumarins; D004247:DNA; D014307:Trioxsalen