Dpb11 Protein Helps Control Assembly of the Cdc45·Mcm2-7·GINS Replication Fork Helicase (original) (raw)

2015, Journal of Biological Chemistry

Background: Dpb11 is required for the initiation of DNA replication. The replication fork helicase is composed of Cdc45, Mcm2-7, and GINS. Results: Dpb11 recruits Cdc45 to Mcm2-7, and Dpb11 blocks GINS interaction with Mcm2-7. Dpb11 also binds to ssDNA, and this interaction releases Dpb11 from Mcm2-7. Conclusion: Dpb11 helps control assembly of the replication fork helicase. Significance: A mechanism for Dpb11 function is described. Dpb11 is required for the initiation of DNA replication in budding yeast. Dpb11 binds to S-phase cyclin-dependent kinase-phosphorylated Sld2 and Sld3 to form a ternary complex during S phase. The replication fork helicase in eukaryotes is composed of Cdc45, Mcm2-7, and GINS. We show here, using purified proteins from budding yeast, that Dpb11 alone binds to Mcm2-7 and that Dpb11 also competes with GINS for binding to Mcm2-7. Furthermore, Dpb11 binds directly to single-stranded DNA (ssDNA), and ssDNA inhibits the Dpb11 interaction with Mcm2-7. We also found that Dpb11 can recruit Cdc45 to Mcm2-7. We identified a mutant of the BRCT4 motif of Dpb11 that remains bound to Mcm2-7 in the presence of ssDNA (dpb11-m1,m2,m3,m5), and this mutant exhibits a DNA replication defect when expressed in budding yeast cells. Expression of this mutant results in increased interaction between Dpb11 and Mcm2-7 during S phase, impaired GINS interaction with Mcm2-7 during S phase, and decreased replication protein A (RPA) interaction with origin DNA during S phase. We propose a model in which Dpb11 first recruits Cdc45 to Mcm2-7. Dpb11, although bound to Cdc45⅐Mcm2-7, can block the interaction between GINS and Mcm2-7. Upon extrusion of ssDNA from the central channel of Mcm2-7, Dpb11 dissociates from Mcm2-7, and Dpb11 binds to ssDNA, thereby allowing GINS to bind to Cdc45⅐Mcm2-7. Finally, we propose that Dpb11 functions with Sld2 and Sld3 to help control the assembly of the replication fork helicase. Dpb11 is required for the initiation of DNA replication in budding yeast (1). The homologs of Dpb11 include Cut5 (Schizosaccharomyces pombe), Mus101 (Xenopus), and TopBP1 (human). In budding yeast, Sld2 (synthetic lethal with dpb11-1) and Sld3 bind to Dpb11 in a cyclin-dependent kinase (CDK)-dependent 2 manner (2-4). Dpb11 (DNA polymerase B-associated protein) binds to CDK-phosphorylated Sld3 and Sld2, and the CDK phosphomimetic mutant of Sld2, Sld2T84D, binds to Dpb11 (2-4). The fusion of Sld3 with Dpb11, when combined with the CDK phosphomimetic mutant of Sld2 (Sld2T84D), bypasses the requirement for CDK in the budding yeast cell (4). These data suggest that formation of the Dpb11-Sld3-Sld2 ternary complex is essential for cell growth. Therefore, Dpb11 is currently viewed as a scaffolding protein for binding Sld2 and Sld3, but the molecular function of Dpb11 in replication initiation is not known. Mcm2-7 forms a hexameric ring, and the subunit composition of this ring is known (Fig. 1B) (5, 6). Mcm2-7 loads onto double-stranded DNA origins during late M phase and G 1 phase. The molecular details of this loading process have been elucidated (7, 8). The Mcm2-7 complex is loaded as a double hexamer, and the Mcm2-7 ring is cracked open at the Mcm2/ Mcm5 interface during this process to encircle doublestranded DNA (9, 10). Mcm2-7 by itself is a very weak ATPase and helicase (5, 6, 11). In S phase, critical events function at the Mcm2-7 complex to activate the replication fork helicase. During S phase, the Dbf4-dependent kinase phosphorylates components of the Mcm2-7 complex, and Cdc45 is recruited to Mcm2-7 (12-14). Cdc45 binds to the Mcm2 subunit of Mcm2-7 (Fig. 1B) (15). Also, during S phase, the Mcm2-7 ring transitions from surrounding double-stranded DNA to encircling single-stranded DNA (15, 16). Therefore, Mcm2-7 cracks open once again in S phase, also at the Mcm2-Mcm5 interface, to allow the extrusion of single-stranded DNA (17). This event is mediated by phosphorylation of Mcm2 by the Dbf4-dependent kinase (17). During S phase, GINS associates with Cdc45⅐Mcm2-7, thereby completing the assembly of the Cdc45⅐Mcm2-7⅐GINS (CMG complex), the replication fork helicase in eukaryotes (18-21). GINS binds to the Mcm3 and Mcm5 subunits of Mcm2-7 (Fig. 1B) (15).