Organization of DNA replication

Abstract

The discovery of the DNA double helix structure half a century ago immediately suggested a mechanism for its duplication by semi-conservative copying of the nucleotide sequence into two DNA daughter strands. Shortly after, a second fundamental step toward the elucidation of the mechanism of DNA replication was taken with the isolation of the first enzyme able to polymerize DNA from a template. In the subsequent years, the basic mechanism of DNA replication and its enzymatic machinery components were elucidated, mostly through genetic approaches and in vitro biochemistry. Most recently, the spatial and temporal organization of the DNA replication process in vivo within the context of chromatin and inside the intact cell are finally beginning to be elucidated. On the one hand, recent advances in genome-wide high throughput techniques are providing a new wave of information on the progression of genome replication at high spatial resolution. On the other hand, novel super-resolution microscopy techniques are just starting to give us the first glimpses of how DNA replication is organized within the context of single intact cells with high spatial resolution. The integration of these data with time lapse microscopy analysis will give us the ability to film and dissect the replication of the genome in situ and in real time.

Figure 1.

Schematic outline of the DNA replication fork and molecular components and enzymatic activities of the replisome. PCNA, proliferating cell nuclear antigen (also termed DNA polymerase clamp or processivity factor). SSB, single strand DNA binding proteins.

Figure 2.

Temporal progression of genome replication. Snapshots of a time-lapse confocal microscopy movie of DNA replication throughout the cell cycle in human HeLa cells stably expressing GFP-tagged PCNA. Times are indicated in hours:minutes and cell cycle phases as G1/SE (S early)/SM (S mid)/SL (S late)/G2/M. Scale bar, 5 µm. During early S phase, small foci distributed throughout the nucleus and mostly corresponding to euchromatic genomic regions are duplicated. Subsequently, the DNA replication machinery loads at perinuclear and perinucleolar heterochromatin regions followed at later times by large constitutive heterochromatic chromosomal regions. This temporal replication program is recapitulated at each cell cycle. See also Movie 1 online at http://cshperspectives.cshlp.org/.

Figure 3.

Model for the progression of genome replication—domino model. (A) One replicon cluster (red) initiates replication at early S phase and DNA synthesis proceeds bidirectionally (replication bubbles). This activates initiation of neighboring replicon clusters (blue), which in turn activate replication at later replicon clusters (green) until the whole chromosome (see scheme below) is fully duplicated in G2 phase. (B) Mouse primary fibroblast cell was pulse labeled for 30 minutes with the nucleotide analogue IdU (early S, red) followed by 3 hours chase, another 30 minutes pulse labeling with CldU (mid S, blue) and 3 hours chase, fixation and detection of replication sites with an antibody to DNA ligase I (late S, green). The micrograph represents a mid optical section of the late S phase fibroblast cell depicting the overlay of the three sequential replication labels and the spatiotemporal progression of genome replication.

Figure 4.

Connecting replicon analysis on DNA fibers to whole cell in situ replication foci. Human HeLa cells expressing GFP-tagged PCNA were imaged using either conventional confocal laser scanning microscopy (CLSM) or super-resolution 3D-structured illumination microscopy (3D-SIM). Shown are maximal intensity projections of the full 3D image stacks illustrating the spatial organization of replication foci in late S phase. The inset shows a twofold magnification of the areas containing replicating heterochromatic regions marked by an asterisk in the image. Scale bar, 2 µm. Even in these condensed heterochromatin domains, the much larger number of individual replicating sites revealed by the increased resolution of the 3D-SIM technique is apparent. Below the cell images, combed DNA fibers are shown with a cartoon representation of nucleotide labeled replicon units overlayed in green. The same replicons are shown underneath with the vertical arrows indicating the origin of replication of each replicon. Several of the replicons are closed together in clusters (underlined by green lines), which initiate replication coordinately. By comparing labeling of replicons in combed DNA fibers and the numbers of replication foci counted by conventional light microscopy, it was concluded that one replication focus corresponds to a spatially organized cluster of replicons, as indicated by the dashed lines. Using 3D-SIM and other super-resolution nanoscopy techniques, much higher numbers of replication foci are resolved. This will soon allow us to measure in detail the characteristics of individual replicons that were formerly visualized only on stretched DNA fibers.