Eukaryotic DNA replication initiates at multiple origins. In early fly and frog embryos, chromosomal replication is very rapid and initiates without sequence specificity. Despite this apparent randomness, the spacing of these numerous initiation sites must be sufficiently regular for the genome to be completely replicated on time. Studies in various eukaryotes have revealed that there is a strict temporal separation of origin "licensing" prior to S phase and origin activation during S phase. This may suggest that replicon size must be already established at the licensing stage. However, recent experiments suggest that a large excess of potential origins are assembled along chromatin during licensing. Thus, a regular replicon size may result from the selection of origins during S phase. We review single molecule analyses of origin activation and other experiments addressing this issue and their general significance for eukaryotic DNA replication.

A strict control of replication origin density and firing time is essential to chromosomal stability. Replication origins in early frog embryos are located at apparently random sequences, are spaced at close (ϳ10-kb) intervals, and are activated in clusters that fire at different times throughout a very brief S phase. Using molecular combing of DNA from sperm nuclei replicating in Xenopus egg extracts, we show that the temporal order of origin firing can be modulated by the nucleocytoplasmic ratio and the checkpoint-abrogating agent caffeine in the absence of external challenge. Increasing the concentration of nuclei in the extract increases S phase length. Contrary to a previous interpretation, this does not result from a change in local origin spacing but from a spreading of the time over which distinct origin clusters fire and from a decrease in replication fork velocity. Caffeine addition or ATR inhibition with a specific neutralizing antibody increases origin firing early in S phase, suggesting that a checkpoint controls the time of origin firing during unperturbed S phase. Furthermore, fork progression is impaired when excess forks are assembled after caffeine treatment. We also show that caffeine allows more early origin firing with low levels of aphidicolin treatment but not higher levels. We propose that a caffeine-sensitive, ATR-dependent checkpoint adjusts the frequency of initiation to the supply of replication factors and optimizes fork density for safe and efficient chromosomal replication during normal S phase.A strict control of replication origin density and time of activation is required to ensure that no DNA stretch is left unreplicated at the end of S phase. Replication initiation is governed by a conserved pathway of protein interactions at DNA replication origins (1). During late mitosis and the G 1 phase, prereplicative complexes are formed at sites defined by ORC, a six-subunit protein complex that directs the loading of other prereplicative complex components, including Cdc6, Cdt1, and the Mcm2-7 complex (2-5). After the G 1 /S transition, the prereplicative complex is converted to a preinitiation complex. This process is triggered by at least two kinases, Cdc7/ Dbf4 and the S-cyclin-dependent kinases, and involves the ordered binding of numerous factors that ultimately unwind origin DNA and recruit DNA polymerases (6, 7).In early Xenopus embryos, S phase is very brief (ϳ20 min), and replication initiates without sequence specificity and at close intervals (ϳ10 kb) (8). Site-specific initiation is only detected after the midblastula transition (MBT), 1 when transcription resumes (9). Replicon size increases slightly at the MBT and more significantly at later stages (9, 10). The mechanisms regulating these changes are unknown, but one clue is that the MBT occurs after a critical number of nuclei accumulate in the embryo (11).A completely random distribution of origins would generate some unacceptably large interorigin distances in the early Xenopus embryo (12). To understand the mechanism...

The subcellular distribution and posttranslational modification of human chromatin assembly factor 1 (CAF-1) have been investigated after UV irradiation of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-associated fraction. This fraction is most abundant during S phase in nonirradiated cells and is much reduced in G2 cells. After UV irradiation, the chromatin-associated form of CAF-1 dramatically increased in all cells irrespective of their position in the cell cycle. Such chromatin recruitment resembles that seen for PCNA, a DNA replication and repair factor. The chromatin-associated fraction of p60 was predominantly hypophosphorylated in nonirradiated G2 cells. UV irradiation resulted in the rapid recruitment to chromatin of phosphorylated forms of the p60 subunit. Furthermore, the amount of the p60 and p150 subunits of CAF-1 associated with chromatin was a function of the dose of UV irradiation. Consistent with these in vivo observations, we found that the amount of CAF-1 required to stimulate nucleosome assembly during the repair of UV photoproducts in vitro depended upon both the number of lesions and the phosphorylation state of CAF-1. The recruitment of CAF-1 to chromatin in response to UV irradiation of human cells described here supports a physiological role for CAF-1 in linking chromatin assembly to DNA repair.