Cdc45 Protein-Single-stranded DNA Interaction Is Important for Stalling the Helicase during Replication Stress

Background: The Dbf4-Cdc7 kinase activates DNA replication, and the helicase is composed of Cdc45, Mcm2-7, and GINS. Results: Dbf4-Cdc7 phosphorylation of Mcm2 is required in vivo for DNA replication, single-stranded DNA accumulation, and GINS-Mcm2-7 interaction. Conclusion: The Dbf4-Cdc7 kinase promotes Mcm2-7 ring opening to allow for origin melting and helicase assembly. Significance: A mechanism for Dbf4-Cdc7 action is described.

show abstract

“…DDK was purified as described (25). Cdc45 was purified as described (31). Protein kinase A was a generous gift from Susan Taylor.…”

Section: Methodsmentioning

confidence: 99%

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

Bruck

Kaplan

2015

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…PKA-Mcm2-7 (PKA tag at the N terminus of Mcm3), GST-Mcm2-7 (GST-tag at the N terminus of Mcm2), and Mcm2-7 were purified as described (26). GST-Pol ⑀ was purified as described (27). Protein kinase A was a generous gift from Susan Taylor.…”

Section: Methodsmentioning

confidence: 99%

“…Antibodies against Psf2 and Cdc45 were generated and purified as described (27). Open Biosystems produced polyclonal antibodies directed against Sld2 or Dpb11 (we supplied the antigens).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The Replication Initiation Protein Sld2 Regulates Helicase Assembly

Bruck

Kaplan

2014

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Background: Sld2 is required for the initiation of DNA replication. Results: An Sld2-Mcm2-7 binding mutant exhibits premature helicase assembly, and an Sld2-DNA binding mutant displays no helicase assembly. Conclusion: Sld2-Mcm interaction is critical to prevent the premature assembly of the replicative helicase, and Sld2-DNA interaction is required for proper helicase assembly. Significance: Sld2 regulates assembly of the replicative helicase.

show abstract

“…Following extensive washes and addition of 50 g/ml Pronase (Calbiochem) to fresh YPGal ϩ doxycycline, cells were further incubated at 37°C for the indicated time. Chromatin immunoprecipitation was performed as described previously (53). We performed PCR with [␣- 32 P]dCTP as a component of the PCR reaction to quantify the amplified product.…”

Section: Methodsmentioning

confidence: 99%

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

Cdc45 Protein-Single-stranded DNA Interaction Is Important for Stalling the Helicase during Replication Stress

Cited by 40 publications

References 54 publications

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

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

The Replication Initiation Protein Sld2 Regulates Helicase Assembly

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

Contact Info

Product

Resources

About