Cardoso's group focused on the dynamics of PCNA, a sliding clamp that aids in DNA polymerase processivity. Recovery of fluorescence after bleaching of PCNA was slow, suggesting that PCNA from within a given replication focus is recycled to load at each new Okazaki fragment.
But the picture over the longer term is very different. Based on pulse labeling of DNA replication and PCNA, bulk PCNA is released from the sites that were replicated earlier and keeps up with the sites of ongoing replication. The recycling at this timescale is indirect—it is unbleached PCNA from the nucleoplasm that assembles at the new replication foci. Importantly, these foci are found preferentially right next to the recently bleached foci.
Earlier workers mapping DNA replication sites had noted, but had not dwelt upon, this close apposition of sequential replication foci. Cardoso believes that the pattern is crucial. “We think the major DNA replication program could be explained by this,” she says. Activity at early replication foci may transmit a signal down the DNA strand to activate neighboring foci, either by pulling physically on the DNA (like undoing a shoelace) or by an unknown chromatin-modifying mechanism. “The program would be fixed,” says Cardoso, “as long as the first sites to fire were fixed.” ▪