Checkpoint-dependent inhibition of DNA replication initiation by Sld3 and Dbf4 phosphorylation

Abstract

SummaryThe initiation of eukaryotic DNA replication is regulated by three protein kinase classes: cyclin dependent kinases (CDK), Dbf4-dependent kinase (DDK) and the DNA damage checkpoint kinases1. CDK phosphorylation of two key initiation factors, Sld2 and Sld3, promotes essential interactions with Dpb112-4 whilst DDK acts by phosphorylating subunits of the Mcm2-7 helicase5. CDK plays an additional role in replication by preventing the re-loading of Mcm2-7 during S, G2 and M phases6, thus preventing origin re-firing and re-replication. During G1-phase, both CDK and DDK are down-regulated, which allows origin licensing and prevents premature replication initiation3. Origin firing is also inhibited during S-phase when DNA damage or replication fork stalling activates the checkpoint kinases7-10. Here we show that, analogous to the situation in G1 phase, the checkpoint kinase Rad53 inhibits both CDK- and DDK-dependent pathways, which acts redundantly to block further origin firing. Rad53 acts on DDK directly by phosphorylating Dbf4, whereas the CDK pathway is blocked by Rad53 phosphorylation of the downstream CDK substrate, Sld3. This allows CDK to remain active during S phase in the presence of DNA damage, which is crucial to prevent reloading of Mcm2-7 onto origins that have already fired6. Our results explain how checkpoints regulate origin firing and demonstrate that the slowing of S phase by the ‘intra-S checkpoint’ is primarily due to the inhibition of origin firing.

Helicase Activation and Establishment of Replication Forks at Chromosomal Origins of Replication

“…Moreover, the Treslin -TopBP1 interaction is reduced in cells treated with hydroxyurea in a checkpoint kinase 1 (Chk1)-dependent manner (Boos et al 2011). This is reminiscent of the situation in which the Rad53 checkpoint kinase phosphorylates Sld3 to inhibit initiation by disrupting the Sld3 -Dpb11 interaction in budding yeast (Lopez-Mosqueda et al 2010;Zegerman and Diffley 2010).…”

“…Under such conditions, further activation of unfired origins is inhibited (Painter and Young 1980;Santocanale and Diffley 1998;Shirahige et al 1998;Larner et al 1999). Studies using budding yeast showed that the checkpoint kinase Rad53 inhibits both the CDK-and DDK-dependent pathways, which are essential for the initiation of replication, by phosphorylating Sld3 and Dbf4, respectively (Lopez-Mosqueda et al 2010;Zegerman and Diffley 2010). Human Treslin/Ticrr, a functional homolog of Sld3, is also inhibited by the phosphorylation checkpoint kinase Chk1 when cells are exposed to DNAreplication stress (Boos et al 2011).…”

The N-terminal 45-kDa Domain of Dna2 Endonuclease/Helicase Targets the Enzyme to Secondary Structure DNA

“…As described under "Materials and Methods," G 1 -arrested cells were released into media containing 20 M CPT or 0.033% MMS. MMS is known to activate the intra-S-phase checkpoint by interfering with progression of replication fork progression (14,47,48), whereas CTP fails to do so (42). DNA was purified from the samples taken at regular (20 or 30 min) intervals after release.…”

A Novel Non-canonical Forkhead-associated (FHA) Domain-binding Interface Mediates the Interaction between Rad53 and Dbf4 Proteins

“…One of the binding partners of FHA1 is Dbf4 (4), the regulatory subunit of the initiator kinase Cdc7 (5,6). The association of Rad53 and Dbf4 mediates the Rad53-dependent phosphorylation of Dbf4 and the consequent inhibition of Cdc7, thus preventing late origin firing (7)(8)(9)(10). This interaction involves the N-terminal region of Dbf4 that folds as a modified BRCA1 C-terminal (BRCT) domain (4,(11)(12)(13).…”