The Dbf4-Cdc7 Kinase Promotes Mcm2-7 Ring Opening to Allow for Single-stranded DNA Extrusion and Helicase Assembly

Bruck, Irina; Kaplan, Daniel L.

doi:10.1074/jbc.m114.608232

Cited by 33 publications

(59 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Antibodies directed against Mcm2, Cdc45, Psf1, and Dpb11 were validated as described previously (25,37,48).…”

Section: Methodsmentioning

confidence: 99%

“…Plasmids for E. coli recombinant expression of Dpb11 (wild-type and mutant) for GINS and for yeast in vivo expression of DPB11 (wild-type and mutants) were prepared as described previously (37,50). Yeast Dpb11 (wild-type and mutant), Mcm2-7, Cdc45, Sld3, Sld2, CDK, DDK, GINS, and GST were purified as described previously (37,46,48,51). Protein kinase A was a generous gift from Susan Taylor (University of California, San Diego, CA).…”

Section: Methodsmentioning

confidence: 99%

“…The kinase was then removed from the mixture by affinity chromatography. GST Pulldown-The GST pulldown assays were performed as described previously (48). GST pulldown reactions were performed in a volume of 100 l and contained GST-tagged protein in GST-binding buffer (40 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 10% glycerol, 0.1% Triton X-100, 1 mM DTT, 0.7 mg/ml pepstatin, 0.1 mM PMSF, and 0.1 mg/ml BSA) and varying amounts of radiolabeled protein as described in each figure.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

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

Bruck

Dhingra

Kaplan

2017

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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...

show abstract

“…Antibodies directed against Mcm2, Cdc45, Psf1, and Dpb11 were validated as described previously (25,37,48).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Bruck

Dhingra

Kaplan

2017

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…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.…”

Section: Dpb11 and Cut5 In Replication Initiationmentioning

confidence: 99%

Multi-BRCT scaffolds use distinct strategies to support genome maintenance

2016

View full text Add to dashboard Cite

Genome maintenance requires coordinated actions of diverse DNA metabolism processes. Scaffolding proteins, such as those containing multiple BRCT domains, can influence these processes by collaborating with numerous partners. The best-studied examples of multi-BRCT scaffolds are the budding yeast Dpb11 and its homologues in other organisms, which regulate DNA replication, repair, and damage checkpoints. Recent studies have shed light on another group of multi-BRCT scaffolds, including Rtt107 in budding yeast and related proteins in other organisms. These proteins also influence several DNA metabolism pathways, though they use strategies unlike those employed by the Dpb11 family of proteins. Yet, at the same time, these 2 classes of multi-BRCT proteins can collaborate under specific situations. This review summarizes recent advances in our understanding of how these multi-BRCT proteins function in distinct manners and how they collaborate, with a focus on Dpb11 and Rtt107.

show abstract

“…35 The 41 Our studies suggesting that DDK phosphorylation of Mcm2 is required for cell growth under normal conditions used a galactose-inducible promoter, wherein galactose levels were varied to achieve wild-type levels of Mcm2 expression under normal conditions. 42 Under these conditions, expression of kinase-dead Mcm2 (Mcm2-S164A, S170A) is lethal to these cells, and overexpression of kinase dead Mcm2 results in a dominant negative severe growth defect. 42 The studies that conclude that DDK phosphorylation of Mcm2 is only important during replication stress were based upon a genomic insertion of the kinasedead mutant of mcm2 at the genomic locus, under regulation by native promoter.…”

Section: Insights Into the Initiation Of Eukaryotic Dna Replicationmentioning

confidence: 99%

Insights into the Initiation of Eukaryotic DNA Replication

Bruck¹,

Pérez-Arnaiz

Colbert

et al. 2015

Nucleus

Self Cite

View full text Add to dashboard Cite

The Dbf4-Cdc7 Kinase Promotes Mcm2-7 Ring Opening to Allow for Single-stranded DNA Extrusion and Helicase Assembly

Cited by 33 publications

References 45 publications

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

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

Multi-BRCT scaffolds use distinct strategies to support genome maintenance

Insights into the Initiation of Eukaryotic DNA Replication

Contact Info

Product

Resources

About