A Positive Amplification Mechanism Involving a Kinase and Replication Initiation Factor Helps Assemble the Replication Fork Helicase

Abstract

Edited by Patrick SungThe assembly of the replication fork helicase during S phase is key to the initiation of DNA replication in eukaryotic cells. One step in this assembly in budding yeast is the association of Cdc45 with the Mcm2-7 heterohexameric ATPase, and a second step is the assembly of the tetrameric GINS (GG-Ichi-Nii-San) complex with Mcm2-7. Dbf4-dependent kinase (DDK) and S-phase cyclin-dependent kinase (S-CDK) are two S phase-specific kinases that phosphorylate replication proteins during S phase, and Dpb11, Sld2, Sld3, Pol ⑀, and Mcm10 are factors that are also required for replication initiation. However, the exact roles of these initiation factors in assembly of the replication fork helicase remain unclear. Key to the initiation of DNA replication is the assembly and activation of the replication fork helicase, the 11-subunit assembly that provides single-stranded DNA templates for the replicative polymerases (1). The replication fork helicase is called Cdc45, GINS (CMG) 3 and is comprised of the Mcm2-7 heterohexameric ATPase and the essential helicase stimulatory factors Cdc45 and GINS (2-5). Cdc45 is a single protein, whereas GINS is a four-subunit complex comprised of Sld5, Psf1, Psf2, and Psf3 (5, 6).The Mcm2-7 complex is a single hexamer when it is free in solution, but when loaded onto DNA in late M phase and G 1 by Orc, Cdc6, and Cdt1 in the presence of ATP, Mcm2-7 forms a double hexamer, with the N termini of the Mcm2-7 proteins forming interhexameric contacts (7,8). In S phase, several changes occur that lead to assembly and activation of the replication fork helicase. Mcm2-7 assembles with Cdc45 and GINS to form the CMG helicase, single-stranded DNA is extruded from the central channel of Mcm2-7 (i.e. origin melting), and the Mcm2-7 double hexamers separate to initiate bidirectional unwinding (8 -11).Five essential DNA replication initiation proteins are also required for replication initiation, and these proteins are Sld2, Sld3, Dpb11, Pol ⑀,. In addition, two S phase-specific kinases, S-CDK and DDK, are required for replication initiation (18 -20). S-CDK phosphorylates Sld2 and Sld3, and phosphorylation of Sld2 and Sld3 results in the formation of a ternary complex with Dpb11 (21,22). In addition, DDK phosphorylates Mcm2, Mcm4, and Mcm6, and DDK phosphorylation of Mcm2-7 is required for cell growth (23)(24)(25).It was shown previously, using an in vitro reconstitution assay, that DDK phosphorylation of Mcm4 and Mcm6 stimulates Sld3 binding to Mcm2-7 with subsequent recruitment of Cdc45 to . GINS recruitment to Mcm2-7 requires all of the initiation factors aside from Mcm10 (27), and GINS binds directly to Dpb11 (28).We report here that Dpb11 stimulates DDK phosphorylation of Mcm4 alone, soluble Mcm2-7, or Mcm2-7 that is loaded onto origin dsDNA. The human homolog of Dpb11, TopBP1, stimulates human DDK phosphorylation of human Mcm4, suggesting that the reaction is conserved from yeast to human. We also find that the DDK phosphomimetic mutant of Mcm4 binds substantially more tightly t...

Cryo-EM structure of Mcm2-7 double hexamer on DNA suggests a lagging-strand DNA extrusion model

“…Likewise, a mutation in the N terminus of Mcm5 (P83L) that also eliminates the requirement for DDK is located in a region of the N terminus of Mcm5 that interacts with Mcm7 and Mcm3, and the mutation could alter the structure of the hexamer–hexamer interface, particularly near the Mcm5 ZnF with its ZFI helix ( 70 ). We suggest that DDK phosphorylation of Mcm4 inactivates the Mcm4-inhibitory domain, and this may be the major factor that triggers the ensuing series of conformational changes that lead to the separation of the two N-tier rings and the dissociation of two hexamers ( 27 , 32 ). Furthermore, because the dsDNA–double hexamer appears to have ADP bound at four of the Mcm2-7 subunit–subunit interfaces and is in an ATPase-inactive state ( 63 ), the conformational changes in the Mcm2-7 during helicase activation may also provide a mechanism for ATP rejuvenation as well as ATPase activation.…”

“…The other two half rings comprised of Mcm2:Mcm4:Mcm6 are separated from each other by a sizable gap and have only a few interactions that bury a small surface of 670 Å 2 . This feature is notable, because Mcm2, Mcm4, and Mcm6, but not Mcm3, Mcm5, and Mcm7, have an extra N-terminal serine/threonine-rich domain (NSD) that inhibits premature replication initiation ( 26 , 27 , 32 , 66 – 69 ). The NSDs of Mcm2, Mcm4, and Mcm6 are not resolved in the cryo-EM structure and thus are disordered.…”

“…At the beginning of S phase, the inactive double hexamer is converted into two active Cdc45–Mcm2-7–GINS (Go, Ichi, Nii, San) (CMG) helicases by the actions of Cdc7-Dbf4–dependent protein kinase (DDK), cyclin-dependent protein kinase (CDK), and an array of protein factors ( 3 , 25 ). Initially DDK-dependent phosphorylation of Mcm2-7 promotes the recruitment of Sld3 and Cdc45 ( 26 – 34 ) before CDK-dependent phosphorylation of Sld2 and Sld3 ( 35 , 36 ) facilitates the binding of a preloading complex consisting of polymerase ε, GINS, Sld2, and Dpb11 ( 37 ). MCM10 binding is required to activate the CMG helicase for the production of ssDNA and splitting of the Mcm2-7 double hexamer ( 38 – 42 ).…”

Single-molecule visualization of Saccharomyces cerevisiae leading-strand synthesis reveals dynamic interaction between MTC and the replisome

“…Replication proteins in S. cerevisiae undergo many posttranslational modifications before and during replication (38,39). For example, Dbf4-dependent kinase (DDK) and cyclindependent kinase (CDK) are known to control replication initiation by phosphorylation of many proteins involved in forming the origin recognition complex (39,40). Additionally, phosphorylation of replisome components plays an important role in programmed fork arrest through phosphorylation of Mcm2-7, which promotes recruitment of Tof1-Csm3 by the replisome (37).…”

“…S. cerevisiae recombinant expressed CMG and Tof1-Csm3 are phosphoproteins that facilitate their interaction (37). Interestingly, upon coexpression of CMG the vast majority of expressed proteins are free Mcm2-7, Cdc45 and GINS that do not simply self-assemble into a CMG complex (40). Thus, it is possible that the small amount of recombinant CMG recovered from expression cells is in fact assembled at origins (2).…”