Visualising chromosomal replication sites and replicons in mammalian cells.
The precise regulation of DNA replication is fundamental to the preservation of intact genomes during cell proliferation. Our understanding of this process has been based traditionally on a combination of techniques including biochemistry, molecular biology and cell biology. In this report we describe how the analysis of the S phase in mammalian cells using classical cell biology techniques has contributed to our understanding of the replication process. We describe traditional and state-of-the-art protocols for imaging sites of DNA synthesis in nuclei and the organisation of active replicons along DNA, as visualised on individual DNA fibres. We evaluate how the different approaches inform our understanding of the replication process, placing particular emphasis on ways in which the higher order chromatin structures and the spatial architecture of replication sites contribute to the orderly activation of defined regions of the genome at precise times of S phase.
University of Manchester, Faculty of Life Sciences, Manchester, UK.
Apolinar
A
Maya-Mendoza
Pedro
P
Olivares-Chauvet
Fanni
F
Kohlmeier
Dean A
DA
Jackson
D000818:Animals; D002875:Chromosomes; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D004261:DNA Replication; D005404:Fixatives / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D005557:Formaldehyde / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D011108:Polymers / Q000737:chemistry; D012093:Replicon; D059807:S Phase Cell Cycle Checkpoints; D059010:Single-Cell Analysis; D013194:Staining and Labeling; D016707:Tissue Fixation
D005404:Fixatives; D011108:Polymers; D005557:Formaldehyde; D004247:DNA; C003043:paraform