Temporal differences in DNA replication during the S phase using single fiber analysis of normal human fibroblasts and glioblastoma T98G cells.
Open Access
- 28 October 2009
- journal article
- Published by Informa UK Limited in Cell Cycle
- Vol. 8 (19), 3133-48
- https://doi.org/10.4161/cc.8.19.9682
Abstract
We have recently shown that replication forks pause near origins in normal human fibroblasts (NHF1-hTERT) but not glioblastoma T98G cells. This observation led us to question whether other differences in the replication program may exist between these cell types that may relate to their genetic integrity. To identify differences, we detected immunoflourescently the sequential incorporation of the nucleotide analogs IdU and CldU into replicating DNA at the start of every hour of a synchronized S phase. We then characterized the patterns of labeled replicating DNA tracks and quantified the percentages and lengths of the tracks found at these hourly intervals. From the directionality of labeling in single extended replicating DNA fibers, tracks were categorized as single bidirectional origins, unidirectional elongations, clusters of origins firing in tandem, or merging forks (terminations). Our analysis showed that the start of S phase is enriched in single bidirectional origins in NHF1-hTERT cells, followed by an increase in clustering during mid S phase and an increase in merging forks during late S phase. Early S phase in T98G cells also largely consisted of single bidirectional origin initiations; however, an increase in clustering was delayed until an hour later, and clusters were shorter in mid/late S phase than in NHF1-hTERT cells. The spike in merging forks also did not occur until an hour later in T98G cells. Our observations suggest models to explain the temporal replication of single and clustered origins, and suggest differences in the replication program in a normal and cancer cell line.Keywords
This publication has 74 references indexed in Scilit:
- A homologous recombination defect affects replication-fork progression in mammalian cellsJournal of Cell Science, 2008
- Spatio‐temporal organization of DNA replication in murine embryonic stem, primary, and immortalized cellsJournal of Cellular Biochemistry, 2005
- Visualization of Altered Replication Dynamics after DNA Damage in Human CellsJournal of Biological Chemistry, 2004
- ATR Homolog Mec1 Promotes Fork Progression, Thus Averting Breaks in Replication Slow ZonesScience, 2002
- Organization of Early and Late Replicating DNA in Human Chromosome TerritoriesExperimental Cell Research, 1999
- Replicon Clusters Are Stable Units of Chromosome Structure: Evidence That Nuclear Organization Contributes to the Efficient Activation and Propagation of S Phase in Human CellsThe Journal of cell biology, 1998
- Chromosome length and DNA loop size during early embryonic development of Xenopus laevisChromosoma, 1993
- The nucleoskeleton and the topology of replicationCell, 1991
- Preferential association of a transcriptionally active gene with the nuclear matrix of rat fibroblasts transformed by a simian-virus-40–pBR322 recombinant plasmidBiochemical Journal, 1990
- Replication origins are attached to the nuclear skeletonNucleic Acids Research, 1986