Activation of mammalian Chk1 during DNA replication arrest

Abstract

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block–induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.

Chk1-dependent slowing of S-phase progression protects DT40 B-lymphoma cells against killing by the nucleoside analogue 5-fluorouracil

“…As far as we are aware this is the first time such a checkpoint response has been documented. The molecular mechanism of replication slowing remains to be determined, however, Chk1 is known to be capable of suppressing replication origin firing (Feijoo et al, 2001;Zachos et al, 2003). This could provide a means of reducing the overall number of replication forks which are active at any one time during S phase in 5FU-treated cells, although effects on replication fork progression per se cannot be excluded on the basis of the current data.…”

“…Previous studies have established that Chk1 is required for multiple replication checkpoint responses including stabilization of stalled replication forks and suppression of latent replication origin firing, with the latter in particular representing a potential mechanism through which DNA synthesis could be blocked or slowed (Feijoo et al, 2001;Zachos et al, 2003). The close correlation between the activation of Chk1 by 5FU in WT DT40 cells and S-phase slowing suggested that the latter might be the result of an active checkpoint process rather than merely inhibition of DNA polymerase activity by dTTP depletion.…”

“…In fission yeast the DNA damage and replication checkpoints are mediated selectively by the Chk1 and Cds1 (the Chk2 homologue) effector kinases, respectively (Rhind and Russell, 2000), however, in vertebrates a substantial body of biochemical and genetic evidence shows that Chk1 is the dominant effector of both pathways (Rhind and Russell, 2000;Bartek et al, 2003;Zachos et al, 2003). In particular, Chk1 is essential for the G2 arrest which delays mitosis in response to DNA damage and to stabilize stalled replication forks and suppress replication origin firing when DNA synthesis is inhibited (Feijoo et al, 2001;Zachos et al, 2003).…”

The destruction box of human Geminin is critical for proliferation and tumor growth in human colon cancer cells

“…Whenever mammalian cells are prevented from completing synthesis from early replicons, ATM (ataxia telangiectasia mutated) independent, checkpoint signal stabilizes components of existing replicons and prevents initiation of replication from late-firing origins (Dimitrova and Gilbert, 2000). Chk1 responds to stalled replication forks as a necessary component for an intra-S phase checkpoint (Feijoo et al, 2001). Thus, one possibility is that DNA damage from incomplete replication initiation is responsible for activating p53 in the À/loxP cells.…”

ATM/ATR-independent inhibition of cyclin B accumulation in response to hydroxyurea in nontransformed cell lines is altered in tumour cell lines

“…Although in all the cell types analysed up to now the checkpoint responses that lead to an intra-S-phase arrest have proved ATR-or Mec1-dependent (Santocanale and Diffley, 1998;Dimitrova and Gilbert, 2000;HekmatNejad et al, 2000;Feijoo et al, 2001), it would be interesting to know whether an ATR-independent response mutated in some cancer cells exists in mammals. This is also relevant to oncogenesis, as uncontrolled firing of origins also induces greater genomic instability (Tanaka and Diffley, 2002).…”