“…However, it was unclear how DDK distinguishes a double hexamer from the single hexamer, as they both contain the same protein components. Recent studies found that DDK interacts with the NTDs of Mcm2 and Mcm4 (Sheu and Stillman 2010;Ramer et al 2013). The architecture of the Mcm2-7 double hexamer, as determined in the present study, now provides a physical explanation for the timed DDK action.…”
Section: Function Of the Mcm2/5 Gate In The Mcm2-7 Double Hexamermentioning
confidence: 69%
“…The Mcm2-7 double-hexamer architecture provides a structural basis for it being a DDK target DDK acts on the Mcm2-7 double hexamer but not the single Mcm2-7 hexamer in solution and in vivo (Sheu and Stillman 2006;Randell et al 2010;Heller et al 2011;Ramer et al 2013;Tanaka and Araki 2013), and the DDK action precedes the S-CDK action to activate the steps toward the actual initiation of DNA synthesis at each origin (Heller et al 2011). However, it was unclear how DDK distinguishes a double hexamer from the single hexamer, as they both contain the same protein components.…”
Section: Function Of the Mcm2/5 Gate In The Mcm2-7 Double Hexamermentioning
Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.
“…However, it was unclear how DDK distinguishes a double hexamer from the single hexamer, as they both contain the same protein components. Recent studies found that DDK interacts with the NTDs of Mcm2 and Mcm4 (Sheu and Stillman 2010;Ramer et al 2013). The architecture of the Mcm2-7 double hexamer, as determined in the present study, now provides a physical explanation for the timed DDK action.…”
Section: Function Of the Mcm2/5 Gate In The Mcm2-7 Double Hexamermentioning
confidence: 69%
“…The Mcm2-7 double-hexamer architecture provides a structural basis for it being a DDK target DDK acts on the Mcm2-7 double hexamer but not the single Mcm2-7 hexamer in solution and in vivo (Sheu and Stillman 2006;Randell et al 2010;Heller et al 2011;Ramer et al 2013;Tanaka and Araki 2013), and the DDK action precedes the S-CDK action to activate the steps toward the actual initiation of DNA synthesis at each origin (Heller et al 2011). However, it was unclear how DDK distinguishes a double hexamer from the single hexamer, as they both contain the same protein components.…”
Section: Function Of the Mcm2/5 Gate In The Mcm2-7 Double Hexamermentioning
Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.
“…p53 (TP53) contributes to CDC6 inhibition 24 , which results in a decreased binding affinity to origin-recognition complexes (ORC), and the formation of the pre-replication complexes (pre-RC), followed by the recruitment of the DNA replicative helicase MCM complex 25 . In addition, the phosphorylation of POLA and MCM protein binding at pre-RC is decreased as a result of the decreased transcript or protein expression of the CDC7/DBF4 complex 26 , and these interaction flows reflect the impairment of normal DNA replication processes.Figure 5Regulatory network model of DNA replication coupled with nicotinamide. ( a ) The colors of the border and inside circle indicate the expression levels of transcriptome and proteome data, respectively.…”
Triple negative breast cancer (TNBC) is characterized by an aggressive biological behavior in the absence of a specific target agent. Nicotinamide has recently been proven to be a novel therapeutic agent for skin tumors in an ONTRAC trial. We performed combinatory transcriptomic and in-depth proteomic analyses to characterize the network of molecular interactions in TNBC cells treated with nicotinamide. The multi-omic profiles revealed that nicotinamide drives significant functional alterations related to major cellular pathways, including the cell cycle, DNA replication, apoptosis and DNA damage repair. We further elaborated the global interaction networks of molecular events via nicotinamide-inducible expression changes at the mRNA and functional protein levels. This approach indicated that nicotinamide treatment rewires interaction networks toward dysfunction in DNA damage repair and away from a pro-growth state in TNBC. To our knowledge, the high-resolution network interactions identified in the present study provide the first evidence to comprehensively support the hypothesis of nicotinamide as a novel therapeutic agent in TNBC.
“…For example, it has been shown in 44 Coupled with the observation that subunits of the Mcm complex are removed from the chromatin via Cdk1 phosphorylation, 46,47 the Cdk1-dependent phosphorylation of Cdc7 on multiple sites prevents Cdc7 from interacting with the chromatin and associated proteins, leading to its removal from origins at G2/M. As cells exit mitosis, they will have to gradually switch back their system to make themselves competent for DNA replication during the next cell division cycle.…”
To maintain genetic stability, the entire mammalian genome must replicate only once per cell cycle. This is largely achieved by strictly regulating the stepwise formation of the pre-replication complex (pre-RC), followed by the activation of individual origins of DNA replication by Cdc7/Dbf4 kinase. However, the mechanism how Cdc7 itself is regulated in the context of cell cycle progression is poorly understood. Here we report that Cdc7 is phosphorylated by a Cdk1-dependent manner during prometaphase on multiple sites, resulting in its dissociation from origins. In contrast, Dbf4 is not removed from origins in prometaphase, nor is it degraded as cells exit mitosis. Our data thus demonstrates that constitutive phosphorylation of Cdc7 at Cdk1 recognition sites, but not the regulation of Dbf4, prevents the initiation of DNA replication in normally cycling cells and under conditions that promote re-replication in G2/M. As cells exit mitosis, PP1a associates with and dephosphorylates Cdc7. Together, our data support a model where Cdc7 (de)phosphorylation is the molecular switch for the activation and inactivation of DNA replication in mitosis, directly connecting Cdc7 and PP1a/Cdk1 to the regulation of once-per-cell cycle DNA replication in mammalian cells.
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