Spatial and Temporal Dynamics of DNA Replication Sites in Mammalian Cells

Abstract

Fluorescence microscopic analysis of newly replicated DNA has revealed discrete granular sites of replication (RS). The average size and number of replication sites from early to mid S-phase suggest that each RS contains numerous replicons clustered together. We are using fluorescence laser scanning confocal microscopy in conjunction with multidimensional image analysis to gain more precise information about RS and their spatial-temporal dynamics. Using a newly improved imaging segmentation program, we report an average of ∼1,100 RS after a 5-min pulse labeling of 3T3 mouse fibroblast cells in early S-phase. Pulse-chase-pulse double labeling experiments reveal that RS take ∼45 min to complete replication. Appropriate calculations suggest that each RS contains an average of 1 mbp of DNA or ∼6 average-sized replicons. Double pulse–double chase experiments demonstrate that the DNA sequences replicated at individual RS are precisely maintained temporally and spatially as the cell progresses through the cell cycle and into subsequent generations. By labeling replicated DNA at the G1/S borders for two consecutive cell generations, we show that the DNA synthesized at early S-phase is replicated at the same time and sites in the next round of replication.

Suppression of replication fork progression in low-dose-specific p53-dependent S-phase DNA damage checkpoint

“…They were then irradiated, incubated for 1 h and pulse labeled for 15 min with CldU to examine the effect of radiation on DNA synthesis (Figure 1b). The staining pattern of replication foci was reported to change during S-phase (Jackson and Pombo, 1998;Ma et al, 1998;Dimitrova and Gilbert, 2000;Leonhardt et al, 2000). Therefore, we focused specifically on cells with the homogeneous IdU staining characteristic to the early S-phase, to examine the changes in the distribution of replication foci 1 h after irradiation.…”

Activation of new replication foci under conditions of replication stress

“…In accordance with previous reports, 2,22 we define early S-phase sub-stage as the period when a large number of small replication foci are detected; these foci are relatively evenly distributed throughout chromatin. Early S lasted for approximately 4.5 h. In mid S-phase replication was active predominantly in foci located under the nuclear envelope and in perinucleolar regions, while less replication foci were seen in other regions of the nucleus.…”

“…The number of distinct replication regions active at any given time is estimated to reach 1100 2 to 1400 in early S-phase, 3 as demonstrated by fluorescence confocal and high resolution microscopy supplemented by appropriate image analysis and deconvolution. One replication focus is thought to contain several active replication forks, 2,4 and the time required to complete replication within this chromatin region is approximately 45 minutes. 2 In early and mid S-phase replication origins are activated in euchromatin.…”

“…One replication focus is thought to contain several active replication forks, 2,4 and the time required to complete replication within this chromatin region is approximately 45 minutes. 2 In early and mid S-phase replication origins are activated in euchromatin. Heterochromatin is replicated in mid and late Sphase.…”

Drosophila JIL-1 kinase mediates histone H3 Ser10 phosphorylation, maintains higher order chromatin structure, and is implicated in dosage compensation

“…This model predicts that regulation of the size of the chromosome surface and the dynamic redistribution of active and inactive genes in the territories are important for transcription regulation. Moreover, it seems that the position of chromatin in the nucleus correlates with the timing of DNA replication (Li et al, 1998;Ma et al, 1998;. In cultured Chinese hamster ovary cells, the early replicating DNA (R band) was found dispersed throughout the nuclear interior, whereas the late replicating DNA (G band) and heterochromatic DNA were found close to the nuclear periphery (Ma et al, 1998).…”