© 2 0 0 3 L a n d e s B i o s c i e n c e . N o t f o r d i s t r i b u t i o n . Undergoing cell division before completing DNA replication would be fatal to any cell. So how do cells know when DNA replication is complete? Over the years, numerous models have been proposed on how this regulation may take place. For example, DNA replication and mitotic entry might be timed solely through the sequential synthesis, activation and degradation of Cyclin/Cdk complexes and their regulators. Such a

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... hanism is sufficient in some cases, such as in the initial divisions of developing Xenopus embryos. However, what would happen if DNA replication were to stall? To accommodate such an event, an additional layer of regulation could exist, one that specifically detects stalled or incomplete DNA replication and responds by inhibiting the timed activation of Cyclin/Cdks. Through groundbreaking genetic studies in yeast and subsequent studies using mammalian cells and Xenopus egg extracts, a signaling pathway that prevents CyclinB/Cdc2 activation in response to stalled DNA replication has been elucidated. 1 The ATR protein kinase and its orthologs in yeast (Rad3 and Mec1) are important regulators of the response to stalled DNA replication. ATR responds to stalled DNA replication by activating Chk1, a conventional protein kinase that interfaces with the normal synchronous regulators of Cdc2 (e.g., Cdc25C and Wee1). These regulators control Cdc2 by modulating phosphorylation of two amino acid residues (T14 and Y15), phosphorylation of which inhibits Cdc2's kinase activity and prevents entrance into M phase. Thus, when DNA replication stalls, the ATR-dependent pathway prevents onset of mitosis by maintaining Cdc2 in an inhibited state through T14/Y15 phosphorylation. Consistent with the importance of this pathway, depletion of ATR from Xenopus extracts 2,3 and deletion of ATR orthologs in yeast 4,5 eliminate the mitotic delay induced by DNA replication inhibitors; in other words, mitosis occurs even though DNA replication remains incomplete. Thus, in these experimental systems and organisms, the ATR-dependent pathway is the predominant regulator of S to M phase transition when DNA replication stalls. Recently however, it has become clear that at least one additional mechanism plays an important role in regulating cell cycle delay in response to stalled DNA replication, at least in intact metazoan cells. Using primary mouse cells in which ATR can be conditionally deleted, we have shown that DNA replication inhibitors prevent entry into mitosis even when ATR is absent. 6 This cell cycle delay occurs even though the canonical pathway that activates Chk1 and maintains inhibitory T14/Y15 phosphorylation of Cdc2 is abolished in ATR knockout cells. Therefore, stalled DNA replication causes inactivation of the CyclinB/Cdc2 complex in ATR knockout cells by some mechanism other than T14/Y15 phosphorylation. The existence of such an ATR-independent mechanism is further supported by studies of Chk1 knockout cells, where the DNA replication checkpoint also remains intact. 7 While there is little doubt that ATR-dependent phosphorylation of Cdc2 on T14/Y15 alone would be sufficient to inhibit mitotic entry, these data imply the existence of at least one other mode of CyclinB/Cdc2 inhibition that operates independently of ATR, Chk1 and Cdc2 T14/Y15 phosphorylation. How does the ATR-independent DNA replication checkpoint work? While the mechanism of regulation is currently an open question, some of the known alternative forms of CyclinB/Cdc2 regulation do not seem to be involved. For example, one way to regulate the CyclinB/Cdc2 complex is through Cyclin B nuclear localization; however, we have shown that stalled DNA replication inhibits M phase entry in ATR knockout cells even when nuclear accumulation of Cyclin B is forced through leptomycin B treatment (Brown & Baltimore, unpublished results). The ATR-independent mechanism also does not appear to require activation of the related kinase, ATM, since the DNA replication checkpoint remains intact even in ATR/ATM double knockout cells. 6 Thus, in primary mouse cells the ATR-independent DNA replication checkpoint seems to operate in a manner that is distinct from the conventional checkpoint pathways first discovered in yeast.