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.

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 (∼10-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.

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DNA polymerase eta (polη) performs translesion synthesis past ultraviolet (UV) photoproducts and is deficient in cancer-prone xeroderma pigmentosum variant (XP-V) syndrome. The slight sensitivity of XP-V cells to UV is dramatically enhanced by low concentrations of caffeine. So far, the biological explanation for this feature remains elusive. Using DNA combing, we showed that translesion synthesis defect leads to a strong reduction in the number of active replication forks and a high proportion of stalled forks in human cells, which contrasts with budding yeast. Moreover, extensive regions of single-strand DNA are formed during replication in irradiated XP-V cells, leading to an over-activation of ATR/Chk1 pathway after low UVC doses. Addition of a low concentration of caffeine post-irradiation, although inefficient to restore S-phase progression, significantly decreases Chk1 activation and abrogates DNA synthesis in XP-V cells. While inhibition of Chk1 activity by UCN-01 prevents UVC-induced S-phase delay in wild-type cells, it aggravates replication defect in XP-V cells by increasing fork stalling. Consequently, UCN-01 sensitizes XP-V cells to UVC as caffeine does. Our findings indicate that polη acts at stalled forks to resume their progression, preventing the requirement for efficient replication checkpoint after low UVC doses. In the absence of polη, Chk1 kinase becomes essential for replication resumption by alternative pathways, via fork stabilization.