Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53

Abstract

Eukaryotic replication origins are licensed by the loading of the replicative DNA helicase, Mcm2-7, in inactive double hexameric form around DNA. Subsequent origin activation is under control of multiple protein kinases that either promote or inhibit origin activation, which is important for genome maintenance. Using the reconstituted budding yeast DNA replication system, we find that the flexible N-terminal tail of Mcm2 promotes the stable recruitment of Dbf4-dependent kinase (DDK) to Mcm2-7 double hexamers, which in turn promotes DDK phosphorylation of Mcm4 and -6 and subsequent origin activation. Conversely, we demonstrate that the checkpoint kinase, Rad53, inhibits DDK binding to Mcm2-7 double hexamers. Unexpectedly, this function is not dependent on Rad53 kinase activity, but requires Rad53 activation by trans-autophosphorylation, suggesting steric inhibition of DDK by activated Rad53. These findings identify critical determinants of the origin activation reaction and uncover a novel mechanism for checkpoint-dependent origin inhibition. Results Residues 1-127 of Mcm2 are dispensable for Mcm2-7 DH stabilityWhile the structured N-terminal domains (NTDs) and AAA+ ATPase domains are conserved between archaeal and eukaryotic MCM proteins, the long unstructured N-terminal tails of Mcm2, -4, and -6 are unique to

Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

“…Figure 1B shows that DDK pre-phosphorylated with Rad53 was unable to promote replication (lane 3), while preincubation of DDK with ATP alone (lane 2) or with ATP and the kinase-dead Rad53 mutant (lane 4) had no effect on replication. Whilst this work was in progress, Wahab and Remus showed that, in their system, binding of Rad53 to Dbf4 is sufficient to inhibit its ability to interact with the MCM double hexamer (Abd Wahab and Remus, 2020). In our hands, the kinase dead Rad53 did not inhibit replication.…”

Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation

“…Figure 1B shows that DDK pre-phosphorylated with Rad53 was unable to promote replication (lane 3), while pre-incubation of DDK with ATP alone (lane 2) or with ATP and the kinase-dead Rad53 mutant (lane 4) had no effect on replication. Whilst this work was in progress, Abd Wahab and Remus also found that Rad53 could directly inhibit DDK action at origins (Abd Wahab and Remus, 2020).…”

“…It has been suggested that Rad53 can inhibit one of its targets, DDK, by direct binding as well as by phosphorylation (Abd Wahab and Remus, 2020). We therefore asked whether we could detect stable interactions between Mrc1 and Rad53 by coimmunoprecipitation using the same buffer conditions used in the in vitro replication assay.…”

Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase

“…In budding yeast however, the checkpoint kinase Rad53 blocks late origin firing, not by inhibition of CDK activity, but by directly inhibiting two replication initiation factors; the DDK subunit Dbf4 and the CDK target Sld3 (Lopez-Mosqueda et al, 2010;Zegerman and Diffley, 2010). Rad53-dependent phosphorylation of Sld3 inhibits its interactions with various other replication factors including Dpb11 (Lopez-Mosqueda et al, 2010;Zegerman and Diffley, 2010), while recent work has shown that direct interaction between Rad53 and Cdc7-Dbf4 prevents DDK from binding to the Mcm2-7 complex (Abd Wahab and Remus, 2020). In higher eukaryotes, in addition to inhibition of CDK activity, the checkpoint can also inhibit origin firing through direct targeting of the Sld3 orthologue Treslin (Boos et al, 2011;Guo et al, 2015) and DDK (Costanzo et al, 2003;Lee et al, 2012).…”