In the absence of the DNA damage checkpoint, treatments that activate the S phase checkpoint kill off (left to right) yeast that lack early firing replication origins (middle row). Adding episomes with early firing origins (p12-Ac) rescues the mutants (bottom row).

Early risers turn on the alarm that prevents genomic instability stemming from problems with DNA replication, say Caldwell et al. The findings show that those DNA replication origins that are activated earliest are needed to trigger the S phase checkpoint.

The S phase checkpoint is turned on during every cell cycle when DNA polymerases stall upon encountering lesions in the DNA template. The checkpoint keeps polymerases on the replication fork, prevents the fork's intricate DNA structure from collapsing, and delays S phase progression into mitosis. In the meantime, the cell has time to find ways around the lesion, for instance by using different polymerases or switching template strands.

In the new work, the authors sought to determine how this checkpoint is activated. They hypothesized that early replication origins might be needed, based on evidence that early origins fire even if cells are not fully ready for replication—if dNTP levels are too low, for example.

Those early origins do not fire in a yeast mutant that lacks a kinase called Dun1 (the basis for the misfiring is so far unknown). The group now shows that, when exposed to treatments that normally activate the S phase checkpoint, dun1 mutants rely on the DNA damage checkpoint for survival. The dependence belies the cells' inability to activate the S phase checkpoint and the resulting damage to the genome that must be repaired before mitosis.

The addition of just one or two functional early origins restored the mutants' S phase checkpoint. The working origins, which were introduced into the yeast cells on episomes, fired normally in the dun1 mutants. The group hypothesizes that these episomal origins might fire because they lack a chromatin structure that somehow cripples endogenous dun1 origins.

Forcing the extra origins to fire later blocked their ability to turn on the S phase checkpoint. So did deletion of their centromeres, suggesting that the checkpoint can be activated by proteins that participate in fork pausing, as occur when forks encounter large protein complexes on centromeres or actively transcribed regions.

Caldwell, J.M., et al.
J. Cell Biol.