Phosphopeptide binding by Sld3 links Dbf4‐dependent kinase to MCM replicative helicase activation

Abstract

The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45‐MCM‐GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin‐dependent kinase (CDK) and Dbf4‐dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK‐dependent manner. Sld3 binds specifically to DDK‐phosphorylated peptides from two MCM subunits (Mcm4, 6) and then recruits Cdc45. MCM mutants that cannot bind Sld3 or Sld3 mutants that cannot bind phospho‐MCM or Cdc45 do not support replication. Moreover, phosphomimicking mutants in Mcm4 and Mcm6 bind Sld3 without DDK and facilitate DDK‐independent replication. Thus, Sld3 is an essential “reader” of DDK phosphorylation, integrating signals from three distinct protein kinase pathways to coordinate DNA replication during S phase.

An Mcm10 Mutant Defective in ssDNA Binding Shows Defects in DNA Replication Initiation

“…In addition, DDK phosphorylates Mcm4 and Mcm6 (22) (23,24) (66) (67) (Figure 8B). DDK phosphorylation of Mcm4 stimulates Sld3 binding to Mcm4 (25) (not shown) and DDK phosphorylation of Mcm4 is required for Sld3 recruitment of Cdc45 to Mcm2-7 (8,21,22,68-70) (35). CDK is also active at this time, catalyzing the phosphorylation of Sld2 and Sld3.…”

“…The essential role of DDK is the phosphorylation of Mcm2-7 (19) (20-22) (23) (24). DDK phosphorylation of Mcm2 reduces the affinity between Mcm2 and Mcm5, which may be important for the opening of the gate to allow the extrusion of the ssDNA from the Mcm2-7 complex (18), while DDK phosphorylation of Mcm4 is required for Cdc45 attachment to Mcm2 (22,25). An mcm5 mutant ( mcm5-P83L ) that bypasses the requirement of DDK in yeast ( mcm5-bob1 mutation) has been described (26).…”

Mcm10 regulates DNA replication elongation by stimulating the CMG replicative helicase

“…As cells progress into S phase, two kinases, S-phase cyclin-dependent kinase (S-CDK) and the Dbf4-dependent Cdc7 kinase (DDK), promote the association of two helicase activators, Cdc45 and GINS, with Mcm2-7. DDK phosphorylation of Mcm2-7 stimulates the association of Cdc45, Sld3, and Sld7 (Heller et al 2011;Deegan et al 2016) followed by the S-CDK-dependent recruitment of a complex between Sld2, Dpb11, DNA polymerase ε (Pol ε), and GINS (Tanaka et al 2007;Zegerman and Diffley 2007;Muramatsu et al 2010;Yeeles et al 2015). Cdc45 and GINS association with Mcm2-7 forms the replicative DNA helicase, the Cdc45/Mcm2-7/GINS (CMG) complex (Moyer et al 2006;Ilves et al 2010), but initial DNA unwinding by this assembly and commitment to replication initiation require Mcm10 (Kanke et al 2012;van Deursen et al 2012;Watase et al 2012).…”

Multi-BRCT scaffolds use distinct strategies to support genome maintenance

“…1). 17,18 Consequently, Dpb11, in conjunction with Sld2 and Sld3, plays a major role in delivering polymerase epsilon and GINS to the origin-loaded MCM, enabling an important intermediate step during replisome assembly.…”

“…Sld3, in complex with Cdc45, recognizes and interacts with MCM that is phosphorylated by DDK (Dbf4-dependent kinase). 16,17 MCM, Cdc45 and GINS form the CMG complex that catalyzes template strand unwinding during replication. 129 In DNA damage checkpoint (right panel), Dpb11 binds 9-1-1 that is located at 5 0 ss-and ds-DNA junction and phosphorylated by Mec1.…”