In eukaryotes, the activation of the prereplicative complex and assembly of an active DNA unwinding complex are critical but poorly understood steps required for the initiation of DNA replication. In this report, we have used bimolecular fluorescence complementation assays in HeLa cells to examine the interactions between Cdc45, Mcm2-7, and the GINS complex (collectively called the CMG complex), which seem to play a key role in the formation and progression of replication forks. Interactions between the CMG components were observed only after the G1/S transition of the cell cycle and were abolished by treatment of cells with either a CDK inhibitor or siRNA against the Cdc7 kinase. Stable association of CMG required all three components of the CMG complex as well as RecQL4, Ctf4/And-1, and Mcm10. Surprisingly, depletion of TopBP1, a homologue of Dpb11 that plays an essential role in the chromatin loading of Cdc45 and GINS in yeast cells, did not significantly affect CMG complex formation. These results suggest that the proteins involved in the assembly of initiation complexes in human cells may differ somewhat from those in yeast systems.T he initiation of eukaryotic DNA replication is a multistep process that requires the assembly of the prereplicative complex (pre-RC), activation of the pre-RC and formation of the replisome (1, 2). Pre-RC assembly occurs during the G 1 phase of the cell cycle by a stepwise recruitment of the origin recognition complex (ORC), Cdc6, Cdt1, and the Mcm2-7 complex onto DNA origins. During the G 1 /S transition stage, the recruitment of other replication factors such as Cdc45 and GINS and the combined actions of two S phase promoting kinases, the cyclindependent (CDK) and Cdc7-Dbf4 (DDK) kinases, lead to the assembly of an active DNA helicase complex at replication origins. This activation results in the unwinding of replication origins, the recruitment of DNA polymerases and accessory factors and the assembly of the replisome for DNA synthesis.In eukaryotic cells, the Mcm2-7 complex seems to be the catalytic core of the replicative helicase. It is essential for origin unwinding and replication fork progression (3-6). It has been shown that a variety of Mcm complexes, including the Mcm4/6/7 subcomplex (from many species), double hexameric complexes of Mcm homologues in Archaea and the budding yeast Mcm2-7 complex, exhibit DNA helicase activity in vitro (1, 7). In vivo, however, formation of an active helicase at replication origins requires the further recruitment of several factors including Cdc45 and GINS, and this activation process is governed by CDK and DDK (1).Both Cdc45 and GINS are required for the establishment and progression of the replication fork (8, 9). These proteins are loaded onto origins during S phase and form a stable complex with Mcm2-7 (10). The complex seems to be a DNA unwinding complex and moves along DNA as part of the replication fork complex (6, 11). Consistent with these observations, a complex of Mcm2-7, Cdc45, and GINS (the CMG complex), purified from D...

Summary Double strand breaks (DSBs) of the DNA in eukaryotic cells are predominantly repaired by non-homologous end joining (NHEJ). The histone chaperone, anti-silencing factor 1a (ASF1a) interacts with MDC1 and is recruited to sites of DSB to facilitate the interaction of phospho-ATM with MDC1 and the phosphorylation of MDC1, which is required for the recruitment of RNF8/RNF168 histone ubiquitin ligases. Thus, ASF1a deficiency reduces histone ubiquitination at DSBs, decreasing the recruitment of 53BP1 and decreases NHEJ, rendering cells more sensitive to DSBs. This role of ASF1a in DSB repair cannot be provided by the closely related ASF1b, and does not require its histone chaperone activity. Homozygous deletion of ASF1a is seen in 10-15% of certain cancers, suggesting that loss of NHEJ may be selected in some malignancies, and that the deletion can be used as a molecular biomarker for cancers susceptible to radiotherapy or to DSB-inducing chemotherapy.

Cdc7 is a serine/threonine kinase that plays essential roles in the initiation of eukaryotic DNA replication and checkpoint response. In previous studies, depletion of Cdc7 by small interfering RNA was shown to induce an abortive S phase that led to the cell cycle arrest in normal human fibroblasts and apoptotic cell death in various cancer cells. Here we report that stressactivated p38 MAP kinase was activated and responsible for apoptotic cell death in Cdc7-depleted HeLa cells. The activation of p38 MAP kinase in the Cdc7-depleted cells was shown to depend on ATR, a major sensor kinase for checkpoint or DNA damage responses. Only the p38 MAP kinase, and not the other stress-activated kinases such as JNK or ERK, was activated, and both caspase 8 and caspase 9 were activated for the induction of apoptosis. Activation of apoptosis in Cdc7-depleted cells was completely abolished in cells treated with small interfering RNA or an inhibitor of the p38 MAP kinase, suggesting that p38 MAP kinase activation was responsible for apoptotic cell death. Taken together, we suggest that the ATR-dependent activation of the p38 MAP kinase is a major signaling pathway that induces apoptotic cell death after depletion of Cdc7 in cancer cells.

Many agents used for chemotherapy, such as doxorubicin, interfere with DNA replication, but the effect of this interference on transcription is largely unknown. Here we show that doxorubicin induces the firing of dense clusters of neoreplication origins that lead to clusters of stalled replication forks in gene-rich parts of the genome, particularly on expressed genes. Genes that overlap with these clusters of stalled forks are actively dechromatinized, unwound, and repressed by an ATR-dependent checkpoint pathway. The ATR checkpoint pathway causes a histone chaperone normally associated with the replication fork, ASF1a, to degrade through a CRL1 bTRCP -dependent ubiquitination/proteasome pathway, leading to the localized dechromatinization and gene repression. Therefore, a globally active checkpoint pathway interacts with local clusters of stalled forks to specifically repress genes in the vicinity of the stalled forks, providing a new mechanism of action of chemotherapy drugs like doxorubicin. Finally, ASF1a-depleted cancer cells are more sensitive to doxorubicin, suggesting that the 7%-10% of prostate adenocarcinomas and adenoid cystic carcinomas reported to have homozygous deletion or significant underexpression of ASF1a should be tested for high sensitivity to doxorubicin.

Though the G(1) checkpoint in mammalian cells has been known for decades, the molecular targets that prevent S-phase entry remain unknown. Mimosine is a rare plant amino acid that arrests the cell cycle in the G(1) phase before entry into S phase. Here, we show that mimosine interrupts the binding of Ctf4 to chromatin, which is essential for the initiation of DNA replication in HeLa cells, and this effect is mediated by the Hif-1α-dependent increase in the level of p27. Depletion of Hif-1α results in an increased binding of Ctf4 to chromatin and the entry of cells into S phase even in the presence of mimosine. These results suggest that the binding of Ctf4 to chromatin is the target of the Hif-1α-dependent checkpoint pathway for cell cycle arrest in G(1) phase. Although we observed Hif-1α-dependent arrest in mimosine-treated cells, it is possible that Ctf4 may act as a common target for G(1) arrest in various other checkpoint pathways.