DBF4, not DRF1, is the crucial regulator of CDC7 kinase at replication forks.
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ABSTRACT:
PROVIDER: PRJNA1046531 | ENA |
REPOSITORIES: ENA
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Project description:In eukaryotes, CDC7 kinase is crucial for DNA replication initiation and has been involved in fork processing and replication stress response. Human CDC7 requires the binding of either one of two regulatory subunits, DBF4 and DRF1, for its activity. However, it is unclear whether the two regulatory subunits target CDC7 to a specific set of substrates, thus having different biological functions, or if they act redundantly. Using genome editing technology, we generated an isogenic set of cell lines deficient in either one of the two CDC7-activating subunits: these cells are viable but present signs of genomic instability, indicating that both DBF4 and DRF1 can independently support CDC7 for bulk DNA replication. Nonetheless, DBF4-deficient cells show altered replication efficiency, including partial deficiency in MCM helicase phosphorylation and alterations in the replication timing of discrete genomic regions. Notably, we find that CDC7 function at replication forks is entirely dependent on DBF4 and not DRF1. Thus, DBF4 is the primary regulator of CDC7 activity, likely mediating most of its functions in unperturbed DNA replication and during replication fork processing upon replication interference.
2024-03-28 | GSE248981 | GEO
Project description:We demonstrate that the Cdc7/Dbf4-dependent histone modification, H3 threonine 45 phosphorylation (H3T45p), is specifically enriched at origins of replication, and highly transcribed genes involved in protein synthesis and glycolysis. Furthermore, we show that H3T45p also mediates polymerase recruitment and expression of these genes.
2022-01-09 | GSE43737 | GEO
Project description:The controlled assembly of replication forks is critical for genome stability. The Dbf4-dependent Cdc7 kinase (DDK) initiates replisome assembly by phosphorylating the MCM2-7 replicative helicase at the N-terminal tails of Mcm2, Mcm4 and Mcm6. At present, it remains poorly understood how DDK docks onto the helicase and how the kinase targets distal Mcm subunits for phosphorylation. By cryo-electron microscopy and biochemical analysis we discovered that an interaction between the HBRCT domain of Dbf4 with Mcm2 serves as an anchoring point, which supports binding of DDK across the MCM2-7 double-hexamer interface and phosphorylation of Mcm4 on the opposite hexamer. Moreover, a rotation of DDK along its anchoring point allows phosphorylation of Mcm2 and Mcm6 on opposite hexamers. In summary, our work provides fundamental insights in the DDK structure, control and the selective activation of the MCM2-7 helicase during DNA replication, which can be exploited for development of novel DDK inhibitors.
2022-05-31 | PXD031315 | Pride
Project description:Centromeres play several important roles in ensuring proper chromosome segregation. Not only do they promote kinetochore assembly for microtubule attachment, but they also support robust sister chromatid cohesion at pericentromeres and facilitate replication of centromeric DNA early in S phase. However, it is still elusive how centromeres orchestrate all these functions at the same site. Here we show that the budding yeast Dbf4-dependent kinase (DDK) accumulates at kinetochores in telophase, facilitated by the Ctf19 kinetochore complex. This promptly recruits Sld3-Sld7 replication initiator proteins to pericentromeric replication origins so that they initiate replication early in S phase. Furthermore DDK at kinetochores independently recruits the Scc2-Scc4 cohesin loader to centromeres in G1 phase. This enhances cohesin loading and facilitates robust pericentromeric cohesion in S phase. Thus, we have found the central mechanism by which kinetochores orchestrate early S phase DNA replication and robust sister chromatid cohesion at microtubule attachment sites. Measurement of genome replication time for various S. cerevisiae strains. For each strain two samples were analysed: a replicating sample (from S phase) and a non-replicating sample (from G2 phase).
2013-06-06 | E-GEOD-41982 | biostudies-arrayexpress
Project description:Centromeres play several important roles in ensuring proper chromosome segregation. Not only do they promote kinetochore assembly for microtubule attachment, but they also support robust sister chromatid cohesion at pericentromeres and facilitate replication of centromeric DNA early in S phase. However, it is still elusive how centromeres orchestrate all these functions at the same site. Here we show that the budding yeast Dbf4-dependent kinase (DDK) accumulates at kinetochores in telophase, facilitated by the Ctf19 kinetochore complex. This promptly recruits Sld3-Sld7 replication initiator proteins to pericentromeric replication origins so that they initiate replication early in S phase. Furthermore DDK at kinetochores independently recruits the Scc2-Scc4 cohesin loader to centromeres in G1 phase. This enhances cohesin loading and facilitates robust pericentromeric cohesion in S phase. Thus, we have found the central mechanism by which kinetochores orchestrate early S phase DNA replication and robust sister chromatid cohesion at microtubule attachment sites.
2013-06-06 | GSE41982 | GEO
Project description:Cdc7 kinase is known to initiate DNA replication, but it is unknown where Cdc7 is found within the genome. We modified the Calling Cards method that uses the Ty5 retrotransposon to investigate Cdc7 binding in the genome. The Ty5 retrotransposon is inserted into the genome by DNA transcription factors or replication factors binding within the genome. We find that Cdc7 inserts Ty5 transposons throughout chromosomes and furthermore creates more Ty5 insertions into regions of DNA that are known to replicate early. Cdc7 does not solely integrate Ty5 at origins or replication, but rather throughout the genome.
2017-09-18 | GSE103943 | GEO
Project description:The regulation of replication forks stalling and replication origins firing must be tightly coordinated to prevents genomic instability. Here we show that GNL3/nucleostemin, a GTP-binding protein able to shuttle between the nucleolus and the nucleoplasm, limits replicative stress by limiting replication origins firing. GNL3 is in proximity of nascent DNA and its depletion reduces forks speed but increases forks density and replication origins firing. When subjected to exogenous replicative stress, cells impaired for GNL3 exhibits MRN-dependent resection and RPA phosphorylation. Strikingly, inhibition of origin firing using CDC7 inhibitor decreased resection in absence of GNL3 but not in absence of BRCA1, suggesting that GNL3 does not protect nascent strand directly from resection. Consistent with this, overexpression of GNL3 leads to an increased resection in response to replicative stress and enhanced origin firing efficiency. These data indicate that the probability of origins firing is tightly regulated by GNL3 to limit replicative stress. Finally, we show that ORC2 and GNL3 interacts together in the nucleolus. We propose a model where GNL3 level is crucial to determine the correct amount of ORC2 on chromatin by sequestrating it in the nucleolus thanks to the capacity of GNL3 to shuttle between nucleoplasm and nucleolus.
2023-07-01 | GSE180865 | GEO
Project description:Centromeres play several important roles in ensuring proper chromosome segregation. Not only do they promote kinetochore assembly for microtubule attachment, but they also support robust sister chromatid cohesion at pericentromeres and facilitate replication of centromeric DNA early in S phase. However, it is still elusive how centromeres orchestrate all these functions at the same site. Here, we show that the budding yeast Dbf4-dependent kinase (DDK) accumulates at kinetochores in telophase, facilitated by the Ctf19 kinetochore complex. This promptly recruits Sld3-Sld7 replication initiator proteins to pericentromeric replication origins so that they initiate replication early in S phase. Furthermore, DDK at kinetochores independently recruits the Scc2-Scc4 cohesin loader to centromeres in G1 phase. This enhances cohesin loading and facilitates robust pericentromeric cohesion in S phase. Thus, we have found the central mechanism by which kinetochores orchestrate early S phase DNA replication and robust sister chromatid cohesion at microtubule attachment sites.
| S-EPMC3679449 | biostudies-literature
Project description:Accurate and efficient DNA synthesis is an essential function of replicating cells. Mutations in replisome components lead to a number of syndromic diseases including immunodeficiencies. Dumbbell former 4 (DBF4) is the regulatory subunit of the DBF4-dependent kinase (DDK) which is essential for the activation of replication origins. We here studied a patient with severe congenital neutropenia (SCN) and syndromic features without a genetic diagnosis. We identified a private homozygous mutation in DBF4 (CADD 25.8 with a DBF4-specific MSC of 3.13) in a patient with SCN. The DBF4 mutant is normally expressed in stimulated PBMCs and dermal fibroblasts, but has a decreased CDC7-binding capacity in overexpression assays. DDK-specific phosphorylation of MCM2 was decreased in stimulated PBMCs with accumulation of CDK inhibitor p21, a G0 cell cycle arrest and impaired proliferation. Serum-starved fibroblasts showed a similar cell cycle phenotype but no p21 accumulation and normal MCM2 phosphorylation. In vitro differentiation of primary CD34+ cells recapitulated the SCN phenotype observed in vivo and was associated with a 4-fold increase in p21 gene expression. Single cell RNA sequencing of whole bone marrow revealed upregulation of p53 targets and activation of the PERK pathway of the unfolded protein response. Autosomal recessive functional DBF4 deficiency causes SCN with syndromic features.
2022-06-10 | GSE160517 | GEO
Project description:DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JMs resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81-Mms4, this cell cycle stage-specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7-Dbf4 (DDK), targets Mus81-Mms4 in conjunction with Cdc5 - both kinases bind to as well as phosphorylate Mus81-Mms4 in an interdependent manner. Moreover, DDK-mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis, establishing DDK as a novel regulator of homologous recombination. The scaffold protein Rtt107, which is part of the Mus81-Mms4 complex, interacts with Cdc7 and thereby targets DDK and Cdc5 to the complex enabling full Mus81 activation. Therefore, Mus81 activation in mitosis involves at least three cell cycle kinases, Cdk1, Cdc5 and DDK. Furthermore, tethering of the kinases in a stable complex with Mus81 is critical for efficient JM resolution
2017-01-21 | PXD005356 | Pride