As a reference sample for free nuclear diffusion, a second plasmid was constructed with m Eos2 fused to a nuclear localization sequence, NLS (GACCCCAAGAAGAAGCGCAAGGTG), which has no other reported biological function.
Using modified nucleotides and fluorescence labeling, these clusters of RF were visualized and found to colocalize with sites of nascent DNA synthesis.
Transition from early to adjacent later replicons within one RF cluster seems to occur by disassembly of PCNA from replication forks into a rapidly diffusing nucleoplasmic pool from where PCNA is recruited to newly activated, nearby replicons were studied at the single-molecule level.
In early S-phase many small clusters of RF are observed throughout the nucleus while in late S-phase fewer but larger clusters of active RF accumulate.
At the molecular level, the assembly of new RFs requires either recycling of PCNA from nearby replication forks or recruitment of PCNA molecules from the nucleoplasmic pool to the replication machinery.
We recorded single-molecule trajectories of PCNA in live cells.
Profiting from the combination of photoactivation and single-molecule tracking, we were able to record large numbers of trajectories per cell.The ratio of these two populations remained constant throughout different stages of S-phase.A fraction of molecules in both populations showed spatially constrained mobility.Here, we present the first single-molecule study on the dynamics of PCNA in replicating and non-replicating nuclei of mammalian cells.We fused PCNA to the photoswitchable protein m Eos2 and generated a cell line stably expressing the construct.For clonal cell line selection, 20 μl of trypsin was applied to divisions containing cell colonies and incubated for 5 minutes.